ABSTRACTLactococcus garvieaeis an emerging zoonotic pathogen, but there are few reports regarding bovine mastitis. The prevalence ofL. garvieaerepresents an increasing disease threat and global public health risk. Thirty-nineL. garvieaeisolates were obtained from 2,899 bovine clinical mastitis milk samples in 6 provinces of China from 2017 to 2021. Five clonal complexes were determined from 32 multilocus sequence types (MLSTs) ofL. garvieae: sequence type 46 (ST46) was the predominant sequence type, and 13 novel MLSTs were identified. All isolates were resistant to chloramphenicol and clindamycin, but susceptible to penicillin, ampicillin, amoxicillin-clavulanic acid, imipenem, ceftiofur, enrofloxacin, and marbofloxacin. Based on genomic analyses,L. garvieaehad 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase. Most isolates hadlsaDandmdtAantimicrobial resistance (AMR) genes. Based on COG (Clusters of Orthologous Genes database) results, the functions of defense, transcription and replication, and recombination and repair were enhanced in unique genes, whereas functions of translation, ribosomal structure, and biogenesis were enhanced in core genes. The KEGG functional categories enriched in unique genes included human disease and membrane transport, whereas COG functional categories enriched in core genes included energy metabolism, nucleotide metabolism, and translation. No gene was significantly associated with host specificity. In addition, analysis of core genome single nucleotide polymorphisms (SNPs) implied potential host adaptation of some isolates in several sequence types. In conclusion, this study characterizedL. garvieaeisolated from mastitis and detected potential adaptations ofL. garvieaeto various hosts.IMPORTANCEThis study provides important genomic insights into a bovine mastitis pathogen,Lactococcus garvieae. Comprehensive genomic analyses ofL. garvieaefrom dairy farms have not been reported. This study is a detailed and comprehensive report of novel features of isolates ofL. garvieae, an important but poorly characterized bacterium, recovered in the past 5 years in 6 Chinese provinces. We documented diverse genetic features, including predominant sequence type ST46 and 13 novel MLSTs.Lactococcus garvieaehad 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase and resistance to chloramphenicol and clindamycin. Most isolates hadlsaDandmdtAantimicrobial resistance genes. However, no gene was significantly associated with host specificity. This is the first report that characterizedL. garvieaeisolates from bovine mastitis and revealed potential host adaptations ofL. garvieaeto various hosts.