The large-scale deployment of Rps (resistance to Phytophthora sojae) genes in soybean has led to the rapid evolution of the virulence profile (pathotype) of P. sojae populations. Determining the pathotypes of P. sojae isolates is important in selecting soybean germplasm carrying the proper Rps, but this process is fastidious and requires specific expertise. In this work, we used a recently developed molecular assay to assess the pathotypes of P. sojae isolates obtained throughout the provinces of Québec, Ontario and Manitoba. In preliminary assays, the molecular tool showed equivalent prediction of the pathotypes as a phenotyping assay and proved to be much faster to apply while eliminating intermediate values. Following the analysis of nearly 300 isolates, 24 different pathotypes were detected in Québec and Ontario, compared to only eight in Manitoba, where soybean culture is more recent. Pathotype 1a, 1c, 1d was predominant in Québec, while 1a, 1b, 1c, 1d, 1k was the most common in Manitoba. Overall, the results showed that 98 and 86% of the isolates carried pathotype 1a or 1c, respectively, suggesting that Rps1a and Rps1c were no longer effective in Canada. Based on the history of soybean varieties used in surveyed fields, it was found that 84% of them contained Rps genes that were no longer resistant against the pathotypes of the isolates found in the fields. While highlighting an easier and more precise option to assess pathotypes, this study presents the first pan-Canadian survey of P. sojae and stresses the importance of carefully managing the declining sources of resistance.