Geographical subspecies of wild silkmoths, Samia cynthia ssp. (Lepidoptera: Saturniidae), differ considerably in sex chromosome constitution owing to sex chromosome fusions with autosomes, which leads to variation in chromosome numbers. We cloned S. cynthia orthologues of 16 Bombyx mori genes and mapped them to chromosome spreads of S. cynthia subspecies by fluorescence in situ hybridization (FISH) to determine the origin of S. cynthia neo-sex chromosomes. FISH mapping revealed that the Z chromosome and chromosome 12 of B. mori correspond to the Z chromosome and an autosome (A₁) of S. c. ricini (Vietnam population, 2n=27, Z0 in female moths), respectively. B. mori chromosome 11 corresponds partly to another autosome (A₂) and partly to a chromosome carrying nucleolar organizer region (NOR) of this subspecies. The NOR chromosome of S. c. ricini is also partly homologous to B. mori chromosome 24. Furthermore, our results revealed that two A₁ homologues each fused with the W and Z chromosomes in a common ancestor of both Japanese subspecies S. c. walkeri (Sapporo population, 2n=26, neo-Wneo-Z) and S. cynthia subsp. indet. (Nagano population, 2n=25, neo-WZ₁Z₂). One homologue, corresponding to the A₂ autosome in S. c. ricini and S. c. walkeri, fused with the W chromosome in S. cynthia subsp. indet. Consequently, the other homologue became a Z₂ chromosome. These results clearly showed a step-by-step evolution of the neo-sex chromosomes by repeated autosome-sex chromosome fusions. We suggest that the rearrangements of sex chromosomes may facilitate divergence of S. cynthia subspecies towards speciation.