A significant feature of the genomes of Lepidoptera, butterflies and moths, is the high conservation of chromosome organization. Recent remarkable progress in genome sequencing of Lepidoptera has revealed that syntenic gene order is extensively conserved across phylogenetically distant species. The ancestral karyotype of Lepidoptera is thought to be n = 31; however, that of the most well studied moth, Bombyx mori, is n = 28, and diverse studies suggest that three chromosomal fusion events occurred in this lineage. To identify the boundaries between predicted ancient fusions involving B. mori chromosomes 11, 23 and 24, we constructed FISH-based chromosome maps of the European corn borer, Ostrinia nubilalis (n = 31). We first determined 511 Mb genomic sequence of the Asian corn borer, Ostrinia furnacalis, a congener of O. nubilalis, and isolated BAC and fosmid clones that were expected to localize in candidate regions for the boundaries using these sequences. Combined with FISH and genetic analysis, we narrowed down the candidate regions to 40 kb-1.5 Mb, in strong agreement with a previous estimate based on the genome of a butterfly, Melitaea cinxia. The significant difference in the lengths of the candidate regions where no functional genes were observed may reflect the evolutionary time after fusion events.