In order to investigate the epidemic and the antibiotic resistance of Streptococcus agalactiae isolated from tilapia (GIFT Oreochromis niloticus) cultured in Huizhou, Zhaoqing, Zhuhai, Zhanjiang cities of Guangdong province, the relationship between beta-lactamase gene and the resistance to penicillin in S. agalactiae was further analyzed. Isolates were identified by amplified specific gene cfb and 16s rDNA; In this study, k-b method was adopted to determine the antimicrobial sensitivity of isolates; the distribution of beta-lactamase gene in strains was detected by PCR assay; and the relationship between beta-lactamase gene and penicillin drugs was analyzed by statistic 6.0. The results showed that the ranking of positive rate of S. agalactiae carried in tilapia was: Huizhou (46.46%) > Zhanjiang (43.24%) > Zhaoqing (17.30%) > Zhuhai (4.17%). Drug sensitive tests showed that all isolates from the four cities were highly resistant to penicillin (with the resistant rate of 94.29%) and sulfamethazine (with the resistant rate of 86.40%), but were sensitive to enrofloxacin (with the resistant rate of only 3.99%). The diversity of the 9 beta-lactamase genes was highly distributed in the isolates. The relevance between the beta-lactamase genes and antibiotic resistance showed that the SAG0658 was significantly correlated with the resistance to ampicillin, suggesting that the SAG0658 might play an important role in the resistance of S. agalactiae to ampicillin. However, there was no significant correlation between the nine beta-lactamase genes and the resistance to penicillin, indicating that there might be other mechanisms underlying the resistance of S. agalactiae to penicillin.