【Aim】 The aim of this study is to isolate microsatellite markers from Tapinoma melanocephalum genome, and to identify polymorphisms of the microsatellite loci. 【Methods】 We developed microsatellite loci from the genomes of 11 geographic populations of T. melanocephalum from the mainland and islands of South China via 454 GS FLX pyrosequencing technology. We used 10 pairs of microsatellite primers selected from randomly designed 100 pairs of microsatellite primers to determine the polymorphisms of the 10 microsatellite loci and to analyze the population genetic diversity and population differentiation of four geographic populations including Dong′ao Island (DAD), Hebao Island (HBD), Meizhou (MZ) and Shanju (SJ) of T. melanocephalum. 【Results】 We successfully developed and isolated 10 pairs of microsatellite primers from the genomes of the 11 geographic populations of T. melanocephalum. In populations DAD, HBD, MZ and SJ, seven of ten microsatellite loci showed high polymorphism and the 10 loci were significantly departed from Hardy-Weinberg equilibrium. The number of allele (A) per locus was 3.50-9.00, allele richness (AR) per locus in each population ranged from 1.992 to 12.938. The AR and expected heterozygosity (HE) from island geographic populations (DAD and HBD) showed no significant difference from those from mainland geographic populations (MZ and SJ). All of the four geographic populations exhibited higher level of genetic divergence (FST=0.15969). HBD and MZ populations showed higher genetic divergence (FST=0.185) and lower gene flow than other paired geographic populations, suggesting that the gene flow of the two populations was restricted. In addition, the genetic variation derived from between individuals within geographic populations. 【Conclusion】 Newly screened microsatellite loci can offer an effective tool for researching on the colony structure and breeding structure of T. melanocephalum, so that its spread mechanism can be deeply understood.