Mastitis is a contagious disease that causes significant milk loss and serious economic damage in dairy animals. Streptococcus agalactiae causes clinical and subclinical mastitis in animals and infections in humans, especially adults, and newborns. This study aimed to confirm the identification of S. agalactiae isolates by PCR in buffalo milk; to determine the antibiotic resistance and virulence genes by multiplex PCR. The material of this study consisted of 30 (12%) S. agalactiae strains identified by phenotypic, biochemical, and PCR methods (rRNA-16S) from 250 isolates with bacterial growth from milk samples of buffaloes with mastitis. In addition, the Kirby-Bauer disc diffusion method was used to test the antibiotic susceptibility of isolates. The antibiotic resistance genes (tetA, tetB, tetK, tetM, tetO, ermA, ermB, ermC, mefA/E, strA, and strB) and the virulence genes (fbsA, hylB, Cluster PI-1, cps C, D, E and cps J, K, e neu B) were investigated by the multiplex PCR method. As a consequence of the study, 13 strains were found to have ermA (43.3%), one strain ermB (3.3%), 15 strains mefA/E (50%), one strain tetO (3.3%), and one strain both tetM and tetK (3.3%). The study conducted in terms of virulence genes showed that 12 strains carried both PI-1 (40%) and cps C, D, E genes (40%), 25 strains had hylB (83.3%), and 13 strains cps J, K, e neu B. Finally, it was concluded that molecular methods are correct and quicker to determine the pathogenic agent, susceptibility and resistance profiles to antibiotics, and the presence of virulence genes at routine intervals in the prevention and control studies for mastitis in farm animals.