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Abstract Soybean (Glycine max (L.) Merr.) has been a commercial oilseed crop in Canada since the 1920s. The production of soybean rapidly increased from 2004 to 2016 in Alberta and Manitoba. Root rot disease caused by the oomycete genus Pythium was observed from 105 fields in 2015 and 2016 in southern Alberta and Manitoba. In the present study, a total of 97 isolates of Pythium were recovered from the root tissues of soybean seedling collected from those fields. Identification of the Pythium species was achieved by examining their morphological features and sequences analysis of internal transcribed spacers (ITS) and cytochrome oxidase subunit 2 gene (Cox II). Nine species of Pythium were identified, with Pythium violae as the predominant species (27 out of 97, 27.8%), followed by P. intermedium (21), P. irregulare (16), P. sylvaticum (12), P. recalcitrans (8), P. ultimum (8), P. heterothallicum (3), P. conidiophorum (1) and P. macrosporum (1). This is the first report of P. conidiophorum , P. recalcitrans and P. violae on soybean in Canada. All the Pythium isolates except three isolates (S2d of P. violae , S29a and S29b of P. heterothallicum) were highly virulent on soybean, causing root rot disease symptoms. Highlights • Obtained 97 Pythium isolates from soybean in Alberta and Manitoba from 2015 to 2016. • Pythium violae was predominant within nine identified Pythium species. • First report of P. conidiophorum , P. recalcitrans and P. violae in soybean in Canada. • All of the Pythium isolates were able to infect soybean with varied aggressiveness. [ABSTRACT FROM AUTHOR]