We investigated interspecific isolating mechanisms and population genetic structure for 19 wild populations of Lilium longiflorum and eight of L. formosanum endemic to the Ryukyu Archipelago and Taiwan. Field observation, artificial interpopulation crosses and isozyme analysis were used. Interspecific isolation was never established by island separation, but it was highly likely by habitat differentiation. There appeared to be additional effects by the unilateral cross incompatibility and isolation in the flowering season. There were significant positive correlation between genetic and geographic distance among populations (r=0.791; p<0.001). Degrees of allozyme variability and divergence at the species level in L. longiflorum were definitely higher than those of L. formosanum. They were extremely high as an insular endemic species. Depauperation of genetic diversity in L. longiflorum populations was associated with islands with lower altitude. This reflects severe bottlenecks by large or complete submergence of such low islands during archipelago development or population rebirth by secondary migration from adjacent islands exempted from large submergence. Combined with historical geography in the archipelago, our investigations suggest that 1) L. formosanum was derived from southern peripheral populations of L. longiflorum by ecological and reproductive isolation, presumably around the late Pleistocene when the mainland of Taiwan was completely separated from the adjacent islands and the continent; and 2) L. longiflorum is an old species in continental islands established around the end of the Pliocene when the current distribution area had been a continuous part of the ancient Asian continent.