A new species of Peronosporales: Phytopythium nanjingense, isolated from the lake water of Nanjing in Jiangsu Province of China is described and illustrated based on morphological characters and supported by molecular phylogenetic analyses using ITS rDNA and Cox1 and Cox2 mtDNA sequences. Phytopythium nanjingense is heterothallic and characterized by aquatic habitat, ellipsoid, mostly obpyriform to ovoid-obpyriform, internally or internally nested proliferating sporangia with conspicuous apical papillae, smooth oogonia, hypogynous antheridia, filamentous antheridial cells, and plerotic or nearly plerotic and thin-walled oospores (0.5–1.6 µm). Differences between the new species and phylogenetically related and morphologically similar species are discussed.