Nine multiplex-PCR-sets, including four triplex and five duplex combinations, were developed from 30 domestic buffalo microsatellite markers based on electrophoresis analysis of PCR products in denaturing polyacrylamide gels and visualization by silver-staining. Further, genetic variability on these microsatellites was examined in Xinglong buffalo of Hainan Province. Twenty-six loci exhibited genetic variation while four microsatellites (CSSM045, ILSTS008, RM099 and HMH1R) were monomorphism. The mean of number of alleles and the expected heterozygosity across 30 microsatellite loci were 4.7 and 0.534, respectively. These optimized multiplex-PCR-sets provide a technical basis for genetic diversity study and parentage test of domestic buffalo.