We analyzed the restriction fragment length polymorphisms in the spacer regions of ribosomal DNA (rDNA), using twelve restriction enzymes, to examine whether the Iriomote cat is related to the leopard cat (Felis bengalensis). A restriction map for each taxon was constructed and the major taxon-specific types of repeating unit (repetypes) were characterized on the basis of the arrangements of restriction sites. The Iriomote cat and the leopard cat share a common repetype but this repetype is different from that of the domestic cat (F. catus) with an estimated sequence divergence of 1.5% and from that of the ocelot (F. paradalis) with an estimated sequence divergence of 2.5%. These results indicate that, phylogenetically, the Iriomote cat is closely related to the leopard cat and that the ancestral population moved from the continent to Iriomote Island quite recently. The rDNA arrays of the leopard cat exhibit considerable intragenomic size-variation, which is thought to have emerged as a result of differences in numbers of repeated DNA segments, whereas the extent of such size-variation is much lower in the rDNA of the Iriomote cat. It appears that, even though migration of the Iriomote cat occurred relatively recently, the population has diverged to some extent from its continental counterpart, perhaps via fixation of preexistent intraspecific variations rather than by generation of new variations.