Abstract Background Pneumonia constitutes a major health challenge in sheep, severely compromising growth rates and overall productivity, and resulting in considerable economic losses to the sheep industry. To address this issue, the development of disease-resistant breeding programs based on the identification of genetic markers associated with pneumonia susceptibility is of critical importance. This study investigated a sheep population on a farm where pneumonia was endemic. The purpose was to use multi-omics methods to rapidly identify the principal pathogens responsible for pneumonia outbreaks, and to screen for genetic loci and key genes related to pneumonia resistance, thereby providing a scientific basis for the implementation of targeted breeding strategies for pneumonia resistance. Results Here, we assessed the impact of pneumonia on sheep growth by evaluating the pneumonia phenotypes of 912 sheep. High-throughput transcriptome sequencing of 40 lungs was conducted to obtain exogenous RNA fragments for microbial sequence alignment. Additionally, 16S rRNA sequencing was performed on lung tissues from 10 healthy and 10 diseased sheep to identify biomarkers associated with phenotypic differences. Mycoplasma ovipneumoniae was identified as the primary pneumonia pathogen, and its presence was further validated by load quantification and immunohistochemical analysis. Integration of genome-wide association study (GWAS) data from 266 lung pathological scores with transcriptome-based differentially expressed genes analysis enabled the identification of five single nucleotide polymorphisms (SNPs) and three potential candidate genes associated with Mycoplasma pneumonia. Subsequent genotyping and phenotype association analyses confirmed the significance of two SNPs and established a strong association between the FOXF1 gene and resistance to Mycoplasma pneumonia. Conclusions High-throughput sequencing technologies have enabled the rapid and accurate identification of the causative pathogen of sheep pneumonia. By integrating multi-omics data, two genomic loci significantly associated with Mycoplasma pneumonia were screened, as well as an anti-Mycoplasma pneumonia key gene, FOXF1. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-025-11699-3. Keywords: Sheep, Multi-omics, Pathogen identification, Mycoplasma ovipneumoniae, Genome-wide association study, Disease-resistant breeding Background Animal diseases are one of the main threats to modern livestock and poultry production. According to reports, in developed countries, economic losses caused by diseases account for about 17% of the total output value of animal husbandry, while in developing countries, this proportion reaches 35–50% [[42]1]. Sheep are economically important livestock species globally. Pneumonia is a respiratory disease that is widely prevalent in sheep farms. The incidence rate of pneumonia in ruminant animals ranges from 10 to 40%, with higher percentages in childhood [[43]2]. Pneumonia can cause growth retardation and increase the risk of secondary pleurisy in sheep, resulting in major production losses [[44]3]. Bacteria-induced infectious pneumonia is most common, and it has been confirmed that pathogens such as Mycoplasma ovipneumoniae, Mycoplasma filamentosa, Pasteurella multocida, Cryptobacterium septicum, Mannheimia haemolytica, Klebsiella pneumoniae, Escherichia coli, Streptococcus, and other pathogens can all cause pneumonia individually or in combination [[45]4–[46]6]. Next generation sequencing (NGS) technology is a promising method for pathogen identification, including rare and newly identified viruses [[47]7]. NGS is independent of cultivation and can efficiently and unbiasedly detect pathogenic microorganisms in clinical samples, providing comprehensive analyses [[48]8, [49]9]. Previous studies have generally focused on pulmonary microbiota composition in healthy sheep [[50]10, [51]11]; while the data on the pulmonary microbiota related to diseases in sheep remains relatively scarce [[52]6]. In microbiome research, comparing 16S rRNA gene sequences has become a reliable approach for identifying microorganisms associated with pathogenicity and infection [[53]12]. Compared to pathogen isolation and identification in veterinary medicine, comprehensive analysis of multi-omics can help to quickly and accurately identify pathogens and discover pathogens that are difficult to cultivate. Mycoplasma ovipneumoniae (M. ovipneumoniae) colonization has been detected in approximately 90% of pneumonia-associated clinical surgery cases [[54]13]. Moreover, it has been identified as the main pathogen causing severe epidemic pneumonia in bighorn sheep [[55]14]. M. ovipneumoniae has also caused severe respiratory disease outbreaks in goats across multiple regions [[56]15, [57]16]. With the increasing proportion of large-scale breeding in recent years, Mycoplasma pneumonia has become the highest incidence rate infectious disease in the sheep industry. The main manifestations of M. ovipneumoniae infections include wheezing, coughing, fever, weight loss, and pulmonary interstitial hyperplasia inflammation [[58]17]. It is characterized by an unusually wide host range, high phenotype, strong infectivity and high incidence rate, and is prone to secondary or mixed infection of other bacteria [[59]18]. Currently, antibiotics remain the main means of treating M. ovipneumoniae infections. However, the long-term use of antibiotics can affect meat quality, produce antibiotic residues, and even lead to the development of drug resistance. Therefore, starting from the genetic basis and screening for resistance genes of Mycoplasma pneumoniae for disease-resistant breeding is an important way to fundamentally address this problem. In farm animal breeding programs, disease resistance and susceptibility—the natural, unique mechanisms by which a host responds to infectious pathogens—are often used as main references. Disease