Objective To screen the polymorphism microsatellite markers of guinea pigs, and provide basic information for identification of genetic structure and gene mapping in guinea pigs. Methods The genomic DNA of guinea pigs was found out on foreign websites, and microsatellite sequences characterized by AC/GT were selected. A total of 400 pairs of primers were designed using software according to their flanking sequences. After two rounds of PCR and electrophoresis, with the electrophoresis bands serving as genetic markers, the polymorphic primers were screened by the evaluation of microsatellite structure of guinea pigs. Results At the first step, 10 DNA samples from 5 strains of guinea pigs were taken for PCR, and 110 pairs of polymorphic microsatellite markers with 1-3 electrophoretic bands were screened from the designed 400 pairs of primers. At the second step, 60 DNA samples from 5 strains of guinea pigs were assayed by PCR and 51 pairs of polymorphic primers with high resolution electrophoresis bands were screened from the above 110 pairs of primers. Among these screened 51 primers, more than 10 pairs of primers showed accordant or mostly accordant differences among strains. Conclusions The 51 pairs of primers are polymorphic among individuals and strains, and have not been found in other reports so far.They can be used for conventional genetic detection and related studies, and some of them can be used for gene mapping of the traits in guinea pigs.