Genetic diversity was studied in four Canadian ecological populations, each corresponding to a Canadian ecozone, of Chondrostereum purpureum, including 93 isolates of various host origin. Pseudo-allelic frequencies were estimated at each of 22 putative RAPD loci by scoring for presence or absence of amplicons in haploid mycelial cultures. The analysis of the hierarchical population structure did not reveal any trend with regard to ecological or host origin. Total gene diversity (HT† = 0.288) was mostly attributable to diversity within populations (HS† = 0.269). In addition, the AMOVA analysis detected most of the molecular variability within subpopulations (89.3%; P < 0.001), whereas a significant (7.3%; P = 0.001) proportion of the gene diversity was found among subpopulations, within ecozones. The results indicate that C. purpureum is a highly heterogeneous pathogen with a continuously distributed population across Canada (GST† = 0.048), and underscore the importance of considering the population structure in the process of its registration as a microbial control agent. A genotype isolated in either central or eastern Canada (ecozones 2, 4 and 5 populations) and selected for its potential as a biophytocide should be considered indigenous in any of these regions of intended use.