Allelic variation in a total of 7 microsatellites was examined between elvers of freshwater eels (Anguilla japonica and Anguilla anguilla). The number of alleles at these loci ranged from 8 to 26. A single test of each locus revealed significant deficits of heterozygotes (P＜0.01). Significant departure from expectations of Hardy-Weinberg equilibrium (HWE) was found for all loci within four subpopulations of A. japonica, which opposes the panmixia hypothesis of Schmidt. Also exact tests of population differentiation based on allelic frequency distribution disagree the hypothesis of random distribution of individuals among populations. Population structure among four populations of A. japonica was revealed with FST value of 0.009 8 (P=0.000 48; 10 000iteration). Pairwise matrixes of FST and RST showed a significant difference between two distantly related species-A. japonica and A. anguilla. Divergent time of the two species calculated by Goldstein method is over 2million years. The results may challenge the Schmidt's theory about the distribution of freshwater eels.