A new species, Pythium subutonaiense, isolated from aquatic environments (lake) in China is being described based on morphological characters and molecular evidence. The isolates grew at temperatures between 5 °C and 38 °C, and the optimum temperature was 30 °C, with a radial growth rate of 17.6 mm at 25 °C per day. It is homothallic and characterized by globose to sub-globose shaped and mostly terminal or sometimes catenulate hyphal swellings, filamentous non-inflated sporangia, and smooth oogonia with hypogynous and monoclinous antheridia that contained one plerotic oospore. In phylogenetic analysis, inferred based on the internal transcribed spacer region of the ribosomal RNA gene and mitochondrial cytochrome c oxidase subunit 1 gene, the new species formed a distinct lineage in Pythium clade B. Differences between the new species and phylogenetically related and morphologically similar species are discussed.