Soybean root and stem rot caused by the oomycete Phytophthora sojae is a destructive disease worldwide that can affect plants at any growth stage. The use of resistant cultivars is the most effective method of controlling the disease. Therefore, monitoring changes in the population of P. sojae regarding the dynamics of avirulence genes capable of overcoming resistance genes (Rps) is important to reduce yield losses and to enhance the effectiveness of the Rps genes. Forty isolates of P. sojae sampled from a region of high incidence of soybean root and stem rot in Brazil were characterized using 14 soybean differentials and 28 pathotypes were identified. Compared to a study conducted a decade ago there was a major shift in pathotype diversity and complexity towards both higher numbers of different pathotypes and of avirulence genes in a given individual in the current population of P. sojae. Breeding programs aiming at developing soybean cultivars with resistance to root and stem rot should consider the high variability in the population of P. sojae and seek for strategic deployment of genes and germplasm.