The signal crayfish Pacifastacus leniusculus, native to northwestern North America, is among the most problematic invaders in freshwater systems of Europe and Japan. To provide an effective tool to analyze population genetics of this alien species, we have developed five polymorphic microsatellite DNA markers from the genome of this species. The number of alleles and expected heterozygosity in each locus ranged between 4–33 and 0.304–0.941, respectively, indicating the utility of the markers in population analysis. We analyzed 212 individuals of P. leniusculus from seven locations in Hokkaido and Honshu, Japan using these markers. When we used a Bayesian clustering analysis, the three clusters, Akashina, Tankai and Hokkaido, were discriminated from each other with different allele frequencies. In addition, a recently found population in Tone River in central Honshu appeared to have originated from Hokkaido, consistent with a previous study using ectosymbiont worms. Thus, the new microsatellite markers are useful in identifying the population structure and genetic connectivity of P. leniusculus in Japan. We discuss potential applications of microsatellite analysis in tracking dispersal pathways and defining eradication units for this invasive crayfish.