Habitat degradation caused by human activities has reduced the sizes of many plant populations worldwide, generally with negative genetic impacts. However, detecting such impacts in tree species is not easy because trees have long life spans. Machilus thunbergii Sieb. et Zucc. (Lauraceae) is a dominant tree species of broad-leaved evergreen forests distributed primarily along the Japanese coast. Inland habitats for this species have become degraded by human activities. To investigate the effects of habitat degradation on genetic structure, we compared the genetic diversities of mature and juvenile trees of five M. thunbergii populations around Lake Biwa in Japan. Allelic diversity was influenced by past lineage admixture events, but the effects of forest size were not clear. On the other hand, the inbreeding coefficient of the juvenile stage was higher in small populations, whereas large populations maintained panmictic breeding. Also, the extent of genetic differentiation was greater in juveniles than in mature trees. We detected the early genetic effects of habitat degradation in small, isolated M. thunbergii populations, indicating that habitat degradation increases inbreeding and genetic differentiation between populations.