Fungal plant pathogens, especially rust fungi (Pucciniales), are well known for their complex life cycles, which include phases of sexual and asexual reproduction. The effect of asexual multiplication on population genetic diversity has been investigated in the poplar rust fungus Melampsora larici-populina using a nested hierarchical sampling scheme. Four hierarchical levels were considered: leaf, twig, tree and site. Both cultivated and wild poplar stands were sampled at two time points at the start and end of rust epidemics. A total of 641 fungal isolates was analyzed using ten microsatellite markers. This study revealed that the genetic signature of asexual multiplication in the wild poplar stand was seen only at lower hierarchical levels (leaf and twig). The observed change in clonal structure, displaying an increase in both gene and genotypic diversity, demonstrates that, despite the fact that only asexual multiplication occurs during the epidemic phase, new genotypes contribute to host infection over time. This genetic analysis provides insights into the balance between auto- and allo-infection processes during the epidemic time course. More generally, this genetic analysis illustrates the utility of population genetics approach for elucidating the proportion of asexual reproduction in the multiplication of isolates during an epidemic, and for proper quantification of asexual dispersal in plant pathogens.