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Streptococcus is a major mastitis-causing pathogen in dairy cows. To investigate the prevalence, antimicrobial resistance and virulence gene of Streptococcus in mastitic milk, a total of 735 mastitic raw milk samples from dairy cows in 11 provinces of China were collected and tested. Antimicrobial resistance of Streptococcus isolates was determined by disc diffusion against 8 classes 29 antimicrobial agents, and Streptococcus resistant genes and virulence genes were determined by PCR and agarose gel electrophoresis. A total of 64 (8.71%) isolates of Streptococcus were isolated and identified using biochemical profiling, including 22 isolates of Streptococcus agalactiae , 13 isolates of Streptococcus dysgalactiae , and 29 isolates of Streptococcus uberis. Out of 64 resistant Streptococcus isolates, all isolates (100%) were resistant to 3 or more antimicrobials. The most frequency (n = 18, 28.12%) of the isolates were multi-resistant to 5–7 antimicrobials and the highest multi-resistant number was 29 (n = 1, 1.56%). Streptococcus isolates had the highest resistance rate to tetracycline (98.44%) and oxacillin (98.44%), followed by penicillin G (96.88%) and doxycycline (96.88%), and the lowest resistance was observed with respect to ciprofloxacin (1.56%). A total of 16 antimicrobials resistance genes with 25 combination patterns were detected in the isolates. The gene combination of Sul1/Sul2/Sul3 + gyrA/parC + cat1/cat2 was the most common pattern (12.5%). The correlation between resistant phenotypes and resistance genes in Streptococcs was 35.87%. A total of 7 virulence genes were detected and 59 (92.19%) isolates harbored at least one gene. Twenty-four classes of gene patterns were found in the isolates and the patterns of bca (9.38%) and cfb (9.38%) were the most prevalent form. In conclusion, the issue of drug resistance of Streptococcus is still a great concern in cattle health in China. • A total of 64 isolates of Streptococcus were isolated from 735 mastitic milk samples from 11 provinces in China. • The consistency between phenotype resistance and resistance genes of Streptococcus were analyzed. • The prevalent virulence genes in Streptococcus were analyzed. [ABSTRACT FROM AUTHOR]