Penicillium nodositatum induces the formation of nodules on the roots of Alnus. The analysis of RFLPs in the PCR-amplified ITS1-5·8S-ITS2 ribosomal sequence demonstrates that all the nodule-inducing strains isolated belong to a homogeneous taxon, P. nodositatum, distinct from other morphologically close species of Penicillium. A gene tree inferred from the ITS1-5·8S-ITS2 PCR/RFLP data shows that the P. nodositatum sequence clusters with those of Penicillium species belonging to subgenus Biverticillium. RAPDs generated using four 10-mer primers, allowed us to identify four distinct groups of strains in a sample of 28 strains of P. nodositatum collected in 10 different geographic sites. Only one group was found in each sampled site. No link could be established between RAPD-based groups and the geographic origin of the strains. However, our results suggest a possible link between these groups and Alnus species and/or habitats associated with each of the four French Alnus species.