The Tsushima leopard cat (Prionailurus bengalensis euptilurus) lives on Tsushima Island in Japan and is a regional population of the Amur leopard cat; it is threatened with extinction. Its genetic management is important because of the small population. We used genotyping by random amplicon sequencing-direct (GRAS-Di) to develop a draft genome and explore single-nucleotide polymorphism (SNP) markers. The SNPs were analyzed using three genotyping methods (mapping de novo, to the Tsushima leopard cat draft genome, and to the domestic cat genome). We examined the genetic diversity and genetic structure of the Tsushima leopard cat. The genome size was approximately 2.435 Gb. The number of SNPs identified was 133–158. The power of these markers was sufficient for individual and parentage identifications. These SNPs can provide useful information about the life of the Tsushima leopard cat and the pairings and for the introduction of founders to conserve genetic diversity with ex situ conservation. We identified that there are no subpopulations of the Tsushima leopard cat. The identifying units will allow for a concentration of efforts for conservation. SNPs can be applied to the analysis of the leopard cat in other regions, making them useful for comparisons among populations and conservation in other small populations.