Objective To develop a multiplex polymerase chain reaction(PCR) method for identification of RR-B strain of Xiphophorus helleri.Methods 6 pairs of special microsatelites primers,consisted of Msa012,Msa014,Msa033,Msb025,Msd003 and Msd051,were selected to perform multiplex PCR to distinguish RR-B swordtail fish strain from wild swordtail fish.Result Stable amplified product with primer series 1(Msa014,Msb025,Msd003) and series 2(Msa012,Msa033,Msd051) could be obtained.Those amplified segments of multiplex PCR could be used to clearly distinguish the strains from each other.The exclusion probability of primer series 1 was 99.98% and the number of primer series 2 was 99.96%.Conclusion The microsatelites multiplex PCR mothed developed in this study can be used to accurately and rapidly identify the RR-B strain of Xiphophorus helleri.