The “Shiba inu” is the most kept and popular among the six Japanese dog breeds (Shiba, Kishu, Shikoku, Hokkaido, Kai, and Akita) officially recognized in Japan, and includes three varieties (two lines of Shinshu-Shiba, San'in-Shiba, and Mino-Shiba) showing different shapes and characters. In the preceding studies on the Japanese dog breeds, only a few studies distinguished these three varieties. Genetic backgrounds of the three varieties are still unclear. In the present study, we revealed genetic diversity and relationship among the three varieties of Shiba by microsatellite markers. We analysed genetic constitutions of the two lines of Shinshu-Shiba (Shinshu-ShibalNippo and Shinshu-Shiba/Shibaho), San'in-Shiba, and Mino-Shiba using 17 microsatellite markers. Shinshu-Shiba was composed of two different societies of preservation, “Nihonken hozonkai” (Nippo) and “Tennenkinenbutsu Shibainu hozonkai” (Shibaho) . In addition, seven dog breeds (Hokkaido, Akita, Shikoku, Satsuma, Ryukyu, Japanese Spitz, and Labrador Retriever) were also analysed by way of comparison. The total number of dogs analysed in a total of 11 populations was 430. Average expected heterozygosities over 17 microsatellite loci of Shinshu-ShibalNippo, Shinshu-Shiba/Shibaho, San'in-Shiba, and Mino-Shiba were 0.641, 0.480, 0.508, and 0.588, respectively. These estimated values were similar as the average (0.603) of the above the seven dog breeds. In addition, the degree of genetic differentiation among the three varieties (including two lines in Shinshu-shiba) of Shiba was highly significant (P<0.01) . A dendrogram was constructed using neighbor-joining (NJ) clustering based on the genetic distances among the 11 populations. The dendrogram exhibited a relatively close relationship among the three varieties (that is, four populations) of Shiba. The San'in-Shiba was distantly separated from both of the Shinshu-Shiba and the Mino-Shiba, while the latter two varieties (that is, three populations) were close to each other. This result might suggest that the ancestor of San'in-Shiba was divergent from those of Shinshu-Shiba (that is, two populations) and Mino-Shiba. We also constructed the other dendrogram based on the genetic distances among individuals belonging to the three varieties (the four populations) of Shiba. All the individuals within each population belonged to the same cluster. This result suggests that each society of preservation of the three varieties (the four populations) of Shiba maintains the pedigree within each population. In this study, we revealed that the degree of genetic differentiation among the three varieties (the four populations) of Shiba was significantly high. From the present analysis, we concluded that the four populations (the two lines of Shinshu-, San'in-, and Mino-) of Shiba should be treated separately from each other in the future study.