Objective To breed a guinea pig inbred strain and set up a method for detection of the microsatellite markers of genetic structure in guinea pigs. Method Using inbreeding methods we try to breed the Zmu-1:DHP inbred strain. With 15 pairs of polymorphism microsatellite primers, the genetic homozygosity of Zmu-1:DHP inbred strain,Zmu-1:DHP outbred strain and Zmu-2:DHP inbred strain (as control) were examined by PCR. Results After breeding for 13 years, 8 sublines of Zmu-1:DHP inbred strain (>20 generations) were bred. After identification, the gene frequency of the second subline of Zmu-1:DHP inbred strain was 86.7%,higher than Zmu-1:DHP outbred strain (6.7%) and Zmu-2:DHP inbred strain (66.7%). The average number of loci of Zmu-1:DHP inbred strain was 1.13,lower than that of Zmu-1:DHP outbred strain (2.47%) and Zmu-2:DHP inbred strain (1.33%). The genotypic frequency of Zmu-1:DHP inbred strain was also higher than that of the other strains. The gene types of Zmu-1:DHP inbred strain were included in the genes of Zmu-1:DHP outbred strain, but Zmu-1:DHP inbred strain was short of 2 characteristic genes. The gene homozygous rates of 8 sublines of Zmu-1:DHP inbred strain were different with each other,among them, those of the 2nd and 8th sublines were higher than others. Conclusions There are both homozygosity and specificity in the Zum-1:DHP inbred strain and Zum-1:DHP outbred strain. The second Zum-1:DHP subline becomes a new inbred strain guinea pig. It is essential that the subline with the characteristic property is screened from these sublines. The guinea pigs of black Zmu-2:DHP inbred strain carrying microsatellite markers not present in the white strains, may carry optimal genes related with hair color properties.