Streptococcus agalactiae, a major bovine mastitis pathogen, poses significant economic and antimicrobial resistance (AMR) challenges. This study evaluated AMR in 128 isolates from Shandong, Hebei and Inner Mongolia using the broth microdilution method. Results showed high sensitivity to most antibiotics (e.g. 100% resistance to penicillin, ceftiofur, amoxicillin, cefquinome and vancomycin) but significant resistance to tetracycline (80.7%) and daptomycin (99.3%). Inner Mongolia isolates exhibited higher resistance and mimimum inhibitory concentrations (MIC) values, reflecting regional antibiotic usage differences, guiding mastitis treatment and antibiotic stewardship in China's dairy industry.