The genetic structure of populations of the sea cucumber Stichopus (Apostichopus) japonicus was investigated using 10 microsatellite markers. In all, 152 individuals from five natural populations were collected from Aomori, Japan (JA and JR), Yosu, South Korea (KY), Dalian, China (CD) and Vladivostock, Russia (RV). A total of 145 alleles were found at 10 loci. The number of alleles per locus ranged from 9 at PSC03 to 20 at SCZ06, with an average of 14.5. Average Ho and He ranged from 0.260 (JR) to 0.434 (JA) and from 0.654 (RV) to 0.778 (KY), respectively. No significant differences at A, Ho and He were found, indicating similar genetic diversity in the five populations. A single allele was found at the PSC05 locus in the RV population. Of the 50 loci, 42 significantly deviated from Hardy–Weinberg equilibrium, all showing heterozygote deficiency. The genetic distances were all relatively great, ranging from 0.497 (between JA and KY) to 1.029 (between KY and JR). This suggests the five populations are genetically distinct. Cluster analysis indicated that JA, KY and CD form one branch and RV and JR another in the UPGMA tree. A hypothesis is proposed for the evolution of the Japanese red sea cucumbers and the genetic relationship among the populations.