In the present study, we have examined the crossability of four species in the A. jaluense complex to provide additional information on their delimitation and levels of differentiation. Crosses between diploid A. chiisanense and the tetraploid species including A. jaluense s. str., A. triphyllum, and A. pseudoproliferum yielded no fruit- or seed-set, indicating that the former species is reproductively completely isolated from the latter three species. In interspecific crosses between the tetraploid species, combinations involving A. jaluense s. str. resulted in much reduced fruit- and seed-set, indicating that A. jaluense s. str. is more or less reproductively isolated from the other tetraploid species. However, crossing data revealed the absence of reproductive isolation between A. triphyllum and A. pseudoproliferum These results strongly support the relationships that have been previously suggested for the A. jaluense complex on the basis of morphology, chromosome number, and flavonoid chemistry.