IntroductionNon-aureusStaphylococcus(NAS) species are currently the most commonly identified microbial agents causing sub-clinical infections of the udder and are also deemed as opportunistic pathogens of clinical mastitis in dairy cattle. More than 10 NAS species have been identified and studied but little is known aboutS. haemolyticusin accordance with dairy mastitis. The present study focused on the molecular epidemiology and genotypic characterization ofS. haemolyticusisolated from dairy cattle milk in Northwest, China.MethodsIn this study, a total of 356 milk samples were collected from large dairy farms in three provinces in Northwest, China. The bacterial isolation and presumptive identification were done by microbiological and biochemical methods following the molecular confirmation by 16S rRNA gene sequencing. The antimicrobial susceptibility testing (AST) was done by Kirby-Bauer disk diffusion assay and antibiotic-resistance genes (ARGs) were identified by PCR. The phylogenetic grouping and sequence typing was done by Pulsed Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) respectively.ResultsIn total, 39/356 (11.0%) were identified as positive forS. haemolyticus. The overall prevalence of otherStaphylococcusspecies was noted to be 39.6% (141/356), while the species distribution was as follows:S. aureus14.9%,S. sciuri10.4%,S. saprophyticus7.6%,S. chromogenes4.2%,S. simulans1.4%, andS. epidermidis1.1%. The antimicrobial susceptibility of 39S. haemolyticusstrains exhibited higher resistance to erythromycin (92.3%) followed by trimethoprim-sulfamethoxazole (51.3%), ciprofloxacin (43.6%), florfenicol (30.8%), cefoxitin (28.2%), and gentamicin (23.1%). All of theS. haemolyticusstrains were susceptible to tetracycline, vancomycin, and linezolid. The overall percentage of multi-drug resistant (MDR)S. haemolyticusstrains was noted to be 46.15% (18/39). Among ARGs,mphCwas identified as predominant (82.05%), followed byermB(33.33%),floR(30.77%),gyrA(30.77%),sul1(28.21%),ermA(23.08%),aadD(12.82%),grlA(12.82%),aacA-aphD (10.26%),sul2(10.26%),dfrA (7.69%), anddfrG (5.13%). The PFGE categorized 39S. haemolyticusstrains into A-H phylogenetic groups while the MLST categorized strains into eight STs with ST8 being the most predominant while other STs identified were ST3, ST11, ST22, ST32, ST19, ST16, and ST7.ConclusionThese findings provided new insights into our understanding of the epidemiology and genetic characteristics ofS. haemolyticusin dairy farms to inform interventions limiting the spread of AMR in dairy production.