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To develop genetic markers for differentiation between pathotypes of the clubroot pathogen Plasmodiophora brassicae, DNA polymorphisms of 85 P. brassicae genes were investigated by comparing the sequences of these genes from published expressed sequence tag libraries to their sequences in the two released whole genomes. A significant portion of the identified sequence differences across all polymorphic genes are between an isolate from New Zealand and the two whole-genome sequenced isolates. Four genes with a high density of polymorphisms were selected and their partial sequences were amplified by polymerase chain reaction (PCR) from the old pathotypes 2, 3, 5, 6, and 8 (based on the Williams differential set) and the new virulent populations 3-like and 5-like. On the sequences of two of the four genes, the old pathotypes are all identical to the two whole-genome sequenced isolates and all of the new virulent populations are identical to the New Zealand isolate. Based on the dimorphism on the sequence of these two genes, an RNase H-dependent PCR protocol was developed. This protocol was demonstrated to be useful for virulent pathotype identification and may also be used to study the population dynamics of P. brassicae and the in planta interaction of different pathotypes.