Abstract Introduction Mycoplasma gallisepticum (MG) infection is a primary cause of chronic respiratory disease in poultry, threatening the economic viability of China’s goose-farming industry. This study investigated the pathogenicity and drug resistance of an MG strain isolated from geese and whole-genome sequenced the strain. Material and Methods A strain designated MG-GD01/22 was isolated from the air-sac tissues of five geese with chronic respiratory disease on a Guangdong goose farm. Its pathogenicity was assessed, antimicrobial susceptibility tests were performed using agar dilution, and its total DNA was extracted for whole-genome sequencing and gene function annotation with second- and third-generation sequencing technologies. The homology of the 16S ribosomal RNA (rRNA) region was analysed and a phylogenetic tree was constructed, as was an evolutionary tree of the mgc2 gene. Gene co-linearity analysis was performed to compare MG-GD01/22 with the strains in the GenBank database. Results The isolate produced “fried egg” colonies and was pathogenic to goslings. It was resistant to enrofloxacin, danofloxacin and spectinomycin and susceptible to valnemulin, tilmicosin, tylosin, acetylisovaleryltylosin tartrate and tiamulin. The genome analysis revealed 1,666 coding genes. Gene database annotation identified 25 virulence-related genes, 22 drug resistance–related genes, 13 pathogen-host–interaction genes and 9 carbohydrate-active enzyme genes. The isolate exhibited 99.9% homology to the MG S6 strain by its 16S rRNA, while the mgc2 gene typing results indicated that it differed from known MG model strains. The genome of MG-GD01/22 showed high homology but poor co-linearity with MG S6, characterised by numerous gene deletions, inversions and displacements. Conclusion This study offers theoretical references for the diagnosis, prevention and treatment of MG in geese in the Guangdong region.