ABSTRACT：Streptococus agalactiae and S.iniae are two common pathogens of fish Streptococcus diseases. Symptoms of disease caused by both Streptococcus pathogens were quite similar, and it was difficult to distinguish the two pathogens from each other directly from the symptoms. Therefore it is necessary to develop a rapid identification method in order to control the disease effectively and rapidly. According to the cfb gene sequence of S. agalactiae and 16S rRNA gene sequence of S. iniae, two pairs of primers were designed and synthesized, and a rapid duplex PCR assay for identification of S. agalactiae and S. iniae was established by optimization of amplification conditions. A 474bp fragment special for S. agalactiae and a 296bp fragment for S. iniae were produced, but no product was amplified from the other common pathogenic bacteria of fish such as Aeromonas hydrophila and Pseudomonas aeruginosa. The method is quite sensitive, and can detect a template concentration as low as 3.2×10-3 ng/μl for S. agalactiae and 3.0×10-2 ng/μl for S. iniae, respectively. A total of eleven pathogen samples collected from pond-cultured tilapia in Guangdong and Hainan provinces were analyzed by duplex PCR. Sequencing and BLAST analysis revealed that all the sequences were cfb gene of S. agalactiae. Molecular identification results were consistent with the previous results of biochemical assays. The double PCR method provides a sensitive and accurate method for rapid identification of S. agalactiae and S. iniae.