The Kurokashiwa breed of native Japanese chickens is primarily reared in Shimane and Yamaguchi Prefectures, Japan. To reveal the genetic diversity and differentiation among the Kurokashiwa populations in both prefectures, 29 microsatellites were analyzed. To prepare the microsatellites, 54, 55, and 24 blood samples were collected from the Kurokashiwa populations of five fancy breeders in Shimane Prefecture, three fancy breeders in Yamaguchi Prefecture, and the Livestock Technology Research Department, Yamaguchi Prefectural Agriculture and Forestry General Technology Center (LTRDY), respectively. The corrected number of alleles (allelic richness: AR) was considerably low (1.63-2.48) through all Shimane and Yamaguchi populations. However, inbreeding coefficient (F_IS) values were not significant in the entire Shimane and Yamaguchi ornamental populations as well as the LTRDY population. In addition, there was no statistically significant difference in expected heterozygosity (H_E) through all populations in Shimane and Yamaguchi Prefectures, although the H_E varied from 0.237 to 0.445 depending on populations. The topology of the phylogenetic tree, pairwise F_ST values, and S_TRUCTURE analysis indicated that Kurokashiwa populations were genetically separated between Shimane and Yamaguchi Prefectures. On the other hand, there was no genetic differentiation among ornamental populations within each prefecture, which seemed to have resulted from adequate random matings by exchanging individuals among fancy breeders within each conservation community, along with a proper random mating within LTRDY. The present study proposes a conservation strategy suggesting that the Kurokashiwa populations of Shimane and Yamaguchi Prefectures should be maintained separately with continuous exchange of birds within each conservation community to preserve genetic diversity. Also, the LTRDY population should be independently kept by avoiding matings with ornamental bird populations because it is a well established closed colony with a uniform genetic constitution.