Taxon delimitation in Pleurotus eryngii species-complex was investigated using an independent loci-based approach. A collection of 47 Pleurotus strains was analyzed through nucleotide sequences of the internal transcribed spacer region (ITS1, 5.8S and ITS2) and M13 minisatellite fragments. A sub-sample made of 12 clones representative of the ITS phylogenetic clusters was sequenced to elucidate variations in the elongation factor 1 alpha gene (EF1-α). A unique clade made of three evolutionary units, i.e. Chinese nebrodensis, Sicilian nebrodensis and eryngii-ferulae from the Mediterranean region, was revealed. The evolutionary genetic divergences were highly correlated (r = 0.86) for both genes. The average number of base substitutions per site was higher for EF1-α gene (0.013) than for ITS (0.001). M13 minisatellite polymorphism marked a clear association between strains and geographic origin but its resolution in taxa delimitation was low. Data from ITS and EF1-α genes supported the hypothesis that the species-complex is a unique gene-pool. Within the species-complex, the nebrodensis type is characterized by a level of nucleotide substitutions lower than as expected for its ranking at a species level.