Objective To assess the genetical structures of three outbred strains of guinea pig and screen the microsatellite loci related to different groups of guinea pigs. Methods Using 45 primers, DNA of three groups of guinea pigs was analyzed by PCR and electrophoresis for genetic markers. Then, the polymorphism of these markers was calculated. In addition, the microsatellite locus related to different hair colors was screened in the three groups of guinea pig. Results All microsatellites loci showed polymorphism. The average number of allelic genes was 2. 29 per locus and the average value of effective genes was 1. 74 per locus. The average expected heterozygosity in total was 0. 39. The average observed heterozygosity was 0. 21. The average PIC was 0. 32, and 68. 9% of the loci showed moderate polymorphism. The average P-value of HW was 0. 1734, but the inheritance in 20 loci of the total locus diverged extremely significantly from the HW balance. The average deviation index D was -0. 407. The differentiation index Fst was 0. 136. The gene flow Nm was 5. 835. Nie genetic similarity of the three groups was low. Conclusions The genetic structures of the three guinea pig groups show a moderate polymorphism. The genetic differences between the groups are not significant. Inheritance of groups diverged from the HW balance and inbreeding within the groups of guinea pigs is obvious. The main reason for these problems is that the number of guinea pigs used for reproduction is low and there are limitations to the approaches of choosing guinea pig seeds. In addition, 12 polymorphic microsatellite loci related to different groups of hair color of guinea pigs and seven specific loci are identified.