To explore genetic differentiation and the genetic relationships of Phytophthora sojae in Anhui Province, the inter-simple sequence repeat (ISSR) technique was used to analyze the genetic diversity of P. sojae. One hundred and sixty ISSR fragments were observed, including 129 (80.6%) polymorphic bands. This suggested that abundant genetic diversity existed among P. sojae in Anhui Province. The pairwise genetic similarity coefficients among the 62 strains ranged from 0.72 to 0.96, with a mean value of 0.85, indicating that there was a high level of genetic variation. Phytophthora sojae strains were divided into five clusters based on neighbor-joining (NJ) analysis, and the clustering was not related to geographic source, according to Mantel’s test (r = 0.3938). In addition, the clustering of strains from the same geographical source had little relationship to the year of collection. Analysis of molecular variance (AMOVA) showed that 16.65% of the genetic variation was derived from the collection area and 83.35% of the genetic variation was within-population variation. The genetic flow between different geographical sources ranged from 0.623 to 2.773, with a mean of 1.325, suggesting that gene exchange was frequent. Genetic distance and the genetic differentiation coefficient were not related to spatial distance.