Isolates of Pythium graminicola and related species were differentiated using restriction fragment length polymorphism (RFLP) analyses of the internal transcribed spacer (ITS) regions of rDNA and the cytochrome c oxidase subunit II (COX II) gene. These sequences were used in subsequent phylogenetic analyses. Finally, the phylogenetic placement of species was compared to that determined from morphological characteristics. The 62 isolates tested were divided into seven groups, A-G, based on RFLP analysis of the rDNA-ITS region. In the RFLP analysis of the COX II gene, isolates were divided into groups similar to those based on ITS-RFLP. Groups A and B were each separated into two additional subgroups. Grouping of isolates based on RFLP analyses agreed with the morphological differentiation. Groups A, B, D, E, F, and G were identified as P. graminicola, P. arrhenomanes, P. aphanidermatum, P. myriotylum, P. torulosum, and P. vanterpoolii, respectively. Group C was closely related to group B based on phylogenetic analysis of the rDNA-ITS region and the COX II gene and is similar to P. arrhenomanes. Each of the other species occupied their own individual clades. Although P. arrhenomanes is morphologically similar to P. graminicola, our phylogenetic analyses revealed that it was evolutionarily distant from P. graminicola and more closely related to P. vanterpoolii. Our analysis also revealed that P. torulosum with smaller oogonia is more closely related to P. myriotylum with large oogonia than to P. vanterpoolii, which forms smaller oogonia and is morphologically similar to P. torulosum. P. aphanidermatum with large oogonia and aplerotic oospores was not related to the morphologically similar species P. myriotylum. Results suggest that P. graminicola and related species are phylogenetically distinct, and molecular analyses, in addition to morphological analyses, are necessary for the accurate taxonomic placement of species in this complex.