Two new diatom species, Praestephanos tokaiensis sp. nov. and P. miensis sp. nov. are described from lacustrine deposits in the Pliocene Ueno Formation of the Kobiwako Group in Iga City, Mie Prefecture, Japan. The stratigraphic ranges of P. tokaiensis and P. miensis are approximately 3.9–3.8 Ma and 3.8–3.7 Ma, respectively. The morphology of these species is mainly characterized by a slightly doubly undulating central area; this feature is not found in previously described Praestephanos species or similar Stephanodiscus species. The stratigraphic distribution and morphology of the two new species suggest that P. tokaiensis and P. miensis have an ancestor–descendant relationship.