Abstract: To analyze molecular typing characteristics of Streptococcus agalactiae isolated from different fish sources and understand the correlation and homology between the strains, this study identified capsular polysaccharide serotype (CPS) with multiplex PCR for 26 S. agalactiae. Multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) were adopted to study the molecular characteristics and homology based on the specific cfb gene of S. agalactiae. The results showed that CPS type had Ia (n=22) and III (n=4) two types, of which S. agalactiae isolated from Schizopygopsis pylzovi and tilapia were Ia, and S. agalactiae isolated from Schizothorax prenati existed Ia (n=2) and III (n=4) two types. Multilocus sequence typing received three STs sequence types (ST-891、ST-103、ST-19), among which the sequence types of S. agalactiae isolated from Schizopygopsis pylzovi and tilapia were new sequence type ST-891, and S. agalactiae isolated from Schizothorax prenati existed ST-19 and ST-103 two sequence types. PFGE cluster analysis created 16 banding patterns (A-P), and the cluster P was the preponderant type. Similar capsular polysaccharide serotype—MLST type strains presented high concentration in the PFGE type. CPS types, MLST types and PFGE types were correlated, but CPS types, STs sequence types and PFGE banding types had no obvious correlation with the host’s sources. The similarity of molecular characteristics of S. agalactiae isolated from different fish sources suggests the possibility of cross infection, and the diversity of molecular characteristics of Schizothorax prenati suggest the existence of heritable variation.