Clubroot, caused by Plasmodiophora brassicae, has become a serious production problem for canola (Brassica napus) on the Canadian prairies. Recently, clubroot-resistant (CR) canola cultivars have begun to show susceptibility to particular strains of P. brassicae. Using next generation sequencing data, a search was undertaken to identify genomic insertions unique to Canadian P. brassicae isolates belonging to two distinct populations. A total of 1353 candidate loci were detected that were present in Canadian P. brassicae isolates and were not homologous to sequences from any other species. Of these candidate loci, 600 appeared to be unique to isolates from the first population, which has been known to be present in Alberta for the longest period of time, while 85 appeared to be unique to isolates from the second population, in which virulence on CR canola cultivars was first detected. From these loci, 11 PCR markers were developed to identify P. brassicae isolates from these two populations. The primers did not amplify DNA from uninfected canola plants, soil samples, bacteria or fungal samples when tested. Assays Pb_pop1_52, Pb_pop1_60, Pb_pop1_255, Pb_pop1_327, Pb_pop1_595, Pb_pop1_673 were specific to population one, whereas primers Pb_pop2_2, Pb_pop2_26, Pb_pop2_33, Pb_pop2_57, and Pb_pop2_94 were specific to population two. The PCR assays were highly sensitive and could detect < 1 pg of target DNA. These methods should be useful for rapid diagnosis and identification of P. brassicae-infected canola plants.