Changes in pathotype structure of Phytophthora sojae populations have been attributed to deployment of race-specific resistant Rps genes in soybean that have been incorporated into commercial cultivars to reduce losses due to Phytophthora root and stem rot. To test this hypothesis, a cultivar rotation study was established from 2007 through 2010 in microplots at a site in Iowa with no history of soybean cultivation. All microplots were inoculated with P. sojae isolate PR1, race 1 (vir 7) prior to planting in year 1. Six rotations were tested: (i) continuous planting of a P. sojae-susceptible cultivar, (ii) continuous planting of a cultivar with high partial resistance to the pathogen, (iii) continuous planting of a cultivar with the Rps 1k gene, (iv) annual rotation of a susceptible with a resistant cultivar, (v) annual rotation of a partially resistant cultivar with a cultivar with the Rps 1k gene, and (vi) 4-year rotation of cultivars with Rps 1k, 1c, 3a, and 1k genes in year one, two, three, and four, respectively. The diversity of 121 isolates of P. sojae that were recovered by baiting from soil samples collected from the experiment were assessed using pathotyping and eight microsatellite markers, and compared with PR1. Changes in pathotype and multilocus genotypes (MLGs) were recorded at the second sampling date, indicating that P. sojae has the ability to evolve quickly. In total, 14 pathotypes and 21 MLGs were recovered over the 4-year experiment, and only 49 and 22% of the isolates had the same pathotype and MLG, respectively, as PR1. The number of isolates of P. sojae recovered varied among rotations, with more isolates recovered from rotations that included a cultivar with partial resistance. Gain of virulence was detected on Rps 1a, 1b, 1c, 1d, and 3a and was not dependent on rotation. Using simple-sequence repeat analysis, 10 alleles that were different from those of PR1 were detected throughout the 4-year period. Cultivar rotation affected the genetic structure of the P. sojae population. Recovery of isolates with different MLGs, genotypic diversity (G = 4.7), and gene diversity (UHe = 0.45) were greater under continuous rotation with partial resistance. Phytophthora root and stem rot causes economic losses in the north-central region of the United States annually. An improved understanding of the effect of Rps gene deployment on P. sojae diversity would lead to improved management practices and reduced losses.