To reexamine the relationships among the Japanese Trillium species that form a polyploid series, we performed principal-coordinates analysis (PCOA) based on proposed karyotypic compositions and on amplified fragment length polymorphism (AFLP) analysis. A hexaploid species, T. smallii, whose karyotypic composition had been hypothesized as K2K2SSUU, with hybridization between tetraploid T. apetalon (SSUU) and a presumed K2K2 diploid species, showed a genotype corresponding to T. yezoense (K1SU). Accordingly, T. smallii appears to be an allopolyploid of T. yezoense, with the karyotypic composition K1K1SSUU. Trillium channellii, a recently described tetraploid species whose origin and genealogical position remains unclear, showed a genotype corresponding to K1K1K2T. We conclude that T. channellii may be derived from hybridization of T. camschatcense (K1K1) as the seed parent and T. tschonoskii (K2K2TT) or hexaploid T. hagae (K1K1K2K2TT) as the pollen parent.