Trueperella pyogenes is considered as a causative agent of many infections, such as mastitis, endometritis, pneumonia, liver abscessation. T. pyogenes can express several virulence genes such as plo, fimA, cbpA, nanH and nanP contributing to its pathogenicity. The aim of this study was to provide an investigation about antimicrobial resistance, as well as virulence genes distribution and gene cassettes among T. pyogenes isolates from dairy cows with clinical mastitis. The susceptibility to different antimicrobial agents was determined by the Broth Microdilution Method, and virulence genes and gene cassette was detected by polymerase chain reactions (PCRs). There are 10.49% (17/162) of milk samples from dairy cows with mastitis were positive for T. pyogenes. High levels of resistance were found to clindamycin (23.53%), oxytetracycline (23.53%), ciprofloxacin (47.06%), sulfamethoxazole/trimethoprim (100%). Moreover, all isolates carried class I integrons, and gene cassette arrays were aadA9 (2/17) or aadA5-dfrA17 (3/17). Finally, all isolates harbored plo nanH and fimA genes, but other genes encoding virulence genes including fimC, fimE, nanP and cbpA are ranged from 47.06% to 88.23%. Our study showed T. pyogenes isolates from dairy cows with clinical mastitis were susceptible to β-lactams. In addition, all seven virulence genes occurred in isolates, and plo, nanH, and fimA gene showed a significantly higher frequency in T. pyogenes of the Liaoning Province, China.