To analyze genetic diversity of endangered medaka (Oryzias latipes), primers for PCR (polymerase chain reaction) were constructed that could amplify ten microsatellite loci of medaka genome based on the contig/scaffold sequences published. DNA was extracted from the caudal fin of medaka collected at six different sampling points in Wanouchi Town, Gifu Prefecture. Genetic diversity within each sampling point was relatively high, whereas genetic differentiation between sampling points was small (pairwise FST= 0.023-0.086) but significant for all except three pairs of the sampling points. The genetic difference between the populations of the sampling points could be explained not only by the geographical distances but by the waterway connectivity between the points. These results indicated the importance of planning agricultural channel corrections considering migration of medaka between populations, to conserve genetic features of medaka in this study site where within-population genetic diversity is not much deteriorated yet.