Forty-seven isolates of Pythium irregulare from different hosts and geographic origins were compared from molecular, morphological and physiological viewpoints. They were divided into four groups (I–IV) based on ITS-RFLP analysis and RAPD analysis. Groups I and II included 32 and eight isolates, respectively, collected from diverse hosts and geographic origins, and groups III and IV comprised seven isolates derived from sugar beet and sugar beet field soil. Group I had smaller oogonia and oospores than did the other three groups. In groups I and II, a significantly higher percentage of the oogonia produced multiple projections compared to groups III and IV which occasionally produced one projection. The growth rate of the four groups was similar at 5–30 °C. At 33°, many isolates of group I grew rapidly but most of the isolates of other groups grew slowly, and at 35°, the former grew but the latter did not. In phylogenetic analysis based on sequences of the ITS region, four groups of P. irregulare were included in one cluster with P. sylvaticum. Groups I–II and III–IV clustered more tightly in the same branch, respectively. The genetic divergence between I–II and III–IV was higher than between each group (I–II and III–IV) and P. sylvaticum, indicating that groups I–II and III–IV may represent two different species.