Aphanomyces root rot of alfalfa, caused by Aphanomyces euteiches, kills seedlings and causes decline of established plants in slowly drained soils. Two races of A. euteiches that are pathogenic to alfalfa have been identified. Despite the contribution of race 1 resistance to establishment and yield of alfalfa, race 1-resistant alfalfa cultivars perform poorly in some fields infested with A. euteiches. Many isolates of A. euteiches obtained from the soils of problematic fields are of a race 2 phenotype. The purpose of this study was to determine distribution, frequency, and pathogenic and genotypic characteristics of race 1 (R1) and race 2 (R2) isolates from 21 fields: 13 in Wisconsin, 7 in Minnesota, and 1 in Kentucky. A. euteiches was successfully isolated from the soil of 16 of the 21 fields; 405 isolates were obtained from Wisconsin, 4 from Minnesota, and 48 from Kentucky. Pathogenicity and race phenotype of isolates were characterized on Saranac (susceptible to R1 and R2 isolates) and WAPH-1 (resistant to R1 and susceptible to R2 isolates) alfalfa populations. One Wisconsin field with no recent history of alfalfa production had a high frequency (51%) of R2 isolates, and 43% of all isolates were R2 from fields with a history of alfalfa production. In a location that was planted continuously to pea for 30 years, 27% of the isolates were R2. Random amplified polymorphic DNA (RAPD) analysis of three R1 and three R2 isolates with eight primers generated 43 total polymorphic bands; however, none of the bands were uniquely associated with race phenotype. Cluster analysis based on RAPD bands revealed no consistent genotypic distinctions between R1 and R2 isolates of A. euteiches. Evaluation of eight commercial alfalfa cultivars for resistance to two R1 and two R2 isolates demonstrated that most are susceptible to R2 isolates; however, those selected for R2 resistance express resistance to R2 isolates. The results suggest that R2 isolates represent a widespread risk to alfalfa cultivars having resistance only to R1 isolates in fields with varied cropping histories.