Abstract: In order to isolate and identify Mycoplasma hyopneumoniae（MHp） from 164 porcine lung tissues suspected of Mycoplasma hyopneumoniae infection collected from several slaughterhouses in Guangdong Province, and to understand the genetic variation characteristics of its main antigen protein genes, the test first carried out 16 S rRNA gene PCR detection on the collected lung tissues, for the positive samples, isolation and culture, morphological observation, duplex PCR identification, sequence analysis on the whole gene of membrane protein P46 and adhesion protein R1 R2 region of P97 of the major antigen protein of the isolated strain, and titer determination were carried out. The results showed that 102 of 164 clinical samples identified by 16 S rRNA were MHp positive, with a positive rate of 62.2%. The positive samples were inoculated into Jiangsu No. 2（KM₂） liquid medium and grew well; the color turned yellow regularly; and specific colony morphology was shown on the solid medium. After duplex RCR identification of 16 S rRNA, the isolated strains of each generation could obtain three target bands with a size of about 1 000 bp. The sequencing results had a nucleotide similarity of 96.42%-98.21% with the reference strains MHp at home and abroad. The results suggested that the three isolates belonged to MHp and could be stably inherited, named JM-24, JM-39, JM-44 respectively. The growth titer of the isolates could reach 1×10^5.67 CCU/mL. The nucleotide similarity of the P46 whole gene and R1 R2 region of P97 gene of the three isolates with the reference strains were 96.0%-99.3% and 97.8%-99.1% respectively. Among them, the ES-2 L strain（accession number CP058578.1） had the highest nucleotide homology between the whole P46 gene and the reference strains, and the strains with the lowest homology were 168（accession number CP002274.1）, 168-L（accession number CP003131.1）, GDZJ01 strains（accession number KF032627.1）; F7.2 C strain（accession number CP034597.1） had the highest nucleotide homology between the R1 R2 region of P97 gene and the reference strains, and the 168/GDZJ01 strain had the lowest homology. The amino acid sequence homology of the R1 R2 region of P97 gene and various pathogenic strains at home and abroad was 72.3%-96.9%, and the amino acid sequence of the isolate JM-44 had deletions or mutations at positions 128-150. The phylogenetic analysis of P46 gene and R1 R2 region of P97 gene showed that the JM-44 isolate was far from the domestic and foreign standard strains and vaccine strains（J strain/168 strain）. The results suggested that the genetic variation between the MHp epidemic strains in Guangdong Province and the current vaccine epidemic strains in the market was large, suggesting that the protection rate of the existing vaccine against Mycoplasma hyopneumoniae may decline.