We examined genetic diversity of the wild Tsushima leopard cat—a regional population of the Amur leopard cat—using microsatellite markers. In addition, we compared genetic diversity of the Tsushima leopard cat with that of the Korean population of Amur leopard cat. Although bias should be considered when applying cross-species amplification, the Tsushima leopard cat showed a lower index of molecular genetic diversity than did the Korean population. These results were consistent with those obtained using other genetic markers, such as mitochondrial DNA and Y chromosome sequences. This low genetic diversity of the wild Tsushima leopard cat may be derived from the founding population. Furthermore, our results suggest that the captive populations held in Japanese zoos may show extremely low genetic diversity, leading to difficulties in genetic management of the Tsushima leopard cat. Moreover, the two regional populations were clearly separated using these marker sets. In the present study, we demonstrated that the genetic diversity of the Tsushima leopard cat is extremely low compared with that of the continental regional population. Importantly, the Japanese captive population for <italic>ex situ</italic> conservation was derived from a founding population with extremely low genetic diversity; hence, we assume that both the captive and wild populations showed extremely low genetic diversities. Our findings emphasize the need to develop carefully considered management strategies for genetic conservation.