Genetic variation and variation in aggressiveness in Phialophora gregata f. sp. sojae, the cause of brown stem rot of soybean, was characterized in a sample of 209 isolates from the north-central region. The isolates were collected from soybean plants without regard to symptoms from randomly selected soybean fields. Seven genotypes (A1, A2, A4, A5, A6, M1, and M2) were distinguished based on DNA fingerprinting with microsatellite probes (CAT)5 and (CAC)5, with only minor genetic variation within the A or M genotypes. Only the A1, A2, and M1 genotypes were represented by more than one isolate. The A genotypes dominated in the eastern Iowa, Illinois, and Ohio samples, whereas the M genotypes were dominant in samples from western Iowa, Minnesota, and Missouri. In growth chamber experiments, isolates segregated into two pathogenicity groups based on their aggressiveness toward soybean cvs. Kenwood and BSR101, which are relatively susceptible and resistant, respectively, to brown stem rot. In both root dip inoculation and inoculation by injecting spores into the stem near the ground line (stab inoculations), isolates of the A genotypes caused greater foliar symptoms and more vascular discoloration than isolates of the M genotypes on both cultivars of soybean. All isolates caused foliar symptoms in both cultivars and in three additional cultivars of soybean with resistance to brown stem rot. Greater differences between the A and M genotypes were seen in foliar symptoms than in the linear extent of xylem discoloration, and greater differences were seen in Kenwood than in BSR101. Inoculation of these genotypes into five cultivars of soybean with different resistance genes to brown stem rot showed a genotype × cultivar interaction. A similar distinction was found in an earlier study of the adzuki bean pathogen, P. gregata f. sp. adzukicola, and consistent with the nomenclature of that pathogen, the soybean pathogens are named the aggressive race (race A) and the mild race (race M) of P. gregata f. sp. sojae.