Volvox rousseletii is a dioecious species belonging to Volvox sect. Volvox that has previously only been found in Africa. During field surveys in a large dam lake (Lake Sagami) in Kanagawa Prefecture, central Japan, we encountered a Volvox sect. Volvox species that produces dioecious sexual spheroids in the water column. Although sexual induction of this species in culture did not produce adequately well-developed sexual spheroids for species identification, molecular data directly obtained from field-collected sexual spheroids verified the identity of field-collected male and female sexual spheroids as well as cultured materials. Based on molecular and morphological data, the species was identified as V. rousseletii. This is the first record of a dioecious species of Volvox sect. Volvox in Japan.