In order to characterize the genetic variation of the poplar pathogen Mycosphaerella populorum (anamorph Septoria musiva), we have studied seven North American populations using the polymerase chain reaction random amplified polymorphic DNA (RAPD) technique. The fungal populations were sampled in 2001 and 2002 by obtaining 352 isolates from cankers and leaf spots in hybrid poplar plantations and adjacent eastern cottonwood (Populus deltoides). A total of 21 polymorphic RAPD markers were obtained with the six RAPD primers used. A fine-level scale analysis of the genetic structure within the populations revealed that subpopulations sampled on P. deltoides and on hybrid trees were not significantly differentiated. In contrast, analyses performed on the entire data set showed high levels of haplotypic diversity and moderate to high genetic differentiation, with 20% of the expected genetic diversity found at the interpopulation level. Moreover, a high and significant correlation between genetic and geographic distances among populations was found, suggesting isolation by distance of the sampled populations. Although the occurrence of the sexual stage of this fungus remained unclear in field populations, five of the six populations were at gametic equilibrium for RAPD loci, suggesting the occurrence of recombination episodes in Septoria musiva populations. Overall, S. musiva appears to consist of differentiated subpopulations, with both asexual and sexual recombination contributing to the local level of genetic structure.