Objective To analyze the population genetics of two outbred colony guinea pigs from two institutions and provide basical information in developing genetic detection methods and standardization of the outbred guinea pigs. Methods 25 polymorphic microsatellite markers were screened by a fluorescent based semi-automated genotyping method for the two populations of guinea pigs, and the population genetic parameters were calculated. Results A total of 121 alleles were detected in the two populations, with 2-10 alleles and a mean 4.84 alleles at each locus. The mean expected heterozygosity was 0.6067, and the average polymorphism information content was 0.552. In the two populations, 103 and 116 alleles were detected, the mean expected heterozygosity was 0.5195 and 0.5838, and mean polymorphism information content was 0.459 and 0.518, respectively. Five loci and six loci, respectively, showed significant deviation from Hardy-Weinberg equilibrium (P<0.05) in the two populations, mostly resulted from heterozygote deficiency. The average Fst of all loci was 0.1056, which implied a moderate genetic differentiation between populations. The Nei' (1972) genetic distance and Nei'(1978) unbiased genetic distance between the two populations were 0.3302 and 0.3204, respectively. Conclusions Both the two populations are consistent with a closed group of animal population genetic characteristics. Several loci deviate from HWE, which probably indicates that a certain degree of inbreeding phenomenon exists during the breeding process.