The identification of plant-associated isolates of Aphanomyces spp. has been mainly based on morphological characters. These types of features, however, can be unreliable due to their high variation and degree of overlap between different taxa. In this work, strains of Aphanomyces, with plant pathogenic and non-pathogenic characteristics, derived mainly from the roots of several leguminous crops in Sweden, as well as a number of reference strains of A. euteiches, A. cladogamus and A. cochlioides were compared by morphology, AT-rich DNA RFLPs and sequence analysis of the total ITS/5.8S rRNA gene region. Parsimony analysis of the sequence data of the total ITS region of 21 strains separated the taxa at the specific level, while two intra-specific groups were also detected within A. euteiches. One comprised all pathogenic isolates of A. euteiches from Sweden, together with two isolates from France and the USA. These isolates formed a homogenous group with identical ITS regions and a single AT-rich DNA RFLP banding pattern. The second ITS subspecific group included reference strains of A. euteiches with preferential pathogenicity to bean and alfalfa, all of which originated from the USA. The bean and alfalfa pathotypes were further distinguished by their different AT-rich DNA RFLPs. The non-pathogenic isolates of Aphanomyces from Sweden associated with the roots of several legumes, formed a genotypically uniform group with an identical ITS sequence and a single AT-rich DNA RFLP pattern. The ITS sequence of this group was identical to that of reference strains of A. cladogamus, suggesting that the non-pathogenic isolates belonged to this species. Intra-specific AT-rich DNA banding profiles were also obtained for isolates of A. cladogamus and these patterns corresponded well to the original hosts.