Objective To screen microsatellite DNA markers from genome of guinea pigs for further genetic quality control and gene-mapping of this species. Methods Microsatellite sequences were obtained by magnetic bead enrichment and genome database screening, and candidate loci were chosen to design primers. Thereafter, genomic DNA of 5 different guinea pig strains were employed to select polymorphic microsatellite DNA markers based on PCR amplification results. Results A total of 304 microsatellite sequences were analyzed by magnetic bead enrichment and 125 primers were designed. One polymorphic microsatellite DNA marker and 17 specific sites (no polymorphic was found) were determined. By gene-mapping, 292 microsatellite sequences were obtained and 178 primers were analyzed, totally 25 polymorphic microsatellite DNA markers and 28 specific sites (without polymorphics) were discovered. Conclusions We obtained 26 polymorphic microsatellite DNA markers and 45 potential markers in guinea pigs, and these may lay a foundation for application of microsatellite DNA markers in genetic quality control and gene-mapping of guinea pigs.