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The delimitation of two ichneumonid species, Spilopteron apicale (Matsumura) and S. tosaense (Uchida), was investigated using DNA sequences of the mitochondrial COI and nuclear 28S rRNA genes, as well as adult morphology. The two species have long been confused and were until recently speculated to be the same species with continuous colour variation. Our molecular and morphological studies reveal that there are two distinct species: a dark species confined to northern or high latitude localities (S. apicale) and a widely distributed species with a marked geographical gradient in body colour (S. tosaense) across the Japanese Archipelago. In the latter species, female body colour became significantly darker with latitude and altitude. A lectotype of Chorischizus apicalis Matsumura, 1912 is designated.