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Streptococcus agalactiae is one of the important causative agents of bovine mastitis. Studies on molecular epidemiology, virulence factors and antimicrobial susceptibility profiles of S. agalactiae isolates from mastitis in China are scarce. Thus, this study was carried out to investigate prevalence of S. agalactiae associated with subclinical mastitis, to determine antimicrobial susceptibility profile, and to analyze their phenotypic and genotypic profiles. Capsular serotypes and genotypes by multilocus serotyping and virulence genes (hemolysin III, C-β protein, C-α protein, surface protein rib, hyaluronate lyase, and C5a peptidase) were determined using molecular assays. Additionally, susceptibility of S. agalactiae isolates to antimicrobial agents was accessed through standard disc diffusion method. A total of 2225 milk samples were collected from 21 large dairy herds located in 10 provinces of China. Overall, 133 (14.1%) S. agalactiae isolates were recovered from 946 (42.5%) subclinical mastitis milk samples. Serotype Ia and ST103 were the most prevalent serotypes and genotypes. Five of the six virulence genes were detected in 22.6% isolates with only two virulence genes and 6.8% of isolates with three genes. There were no significant geographical differences in the distribution of virulence genes. Moreover, 63.9% of S. agalactiae exhibited resistance to levofloxacin, 3.8% isolates were resistant to cefepime, 2.3% were resistant to chloramphenicol and ampicillin. Additionally, two ampicillin/cefotaxime/ levofloxacin-resistant isolates, one vancomycin-resistant isolate and one ampicillin/cefotaxime-resistant isolate were identified. This study concludes high prevalence of S. agalactiae from subclinical mastitis carrying virulence genes and mainly belonging to Serotype Ia and ST103. [ABSTRACT FROM AUTHOR]