Assessment of genetic and phenotypic diversity is necessary to confidently distinguish genotypes of Pleurotus eryngii when seeking traits of interest and to identify strains with high yield potential. We studied 154 strains from Italy for quantitative (shape, size and yield), qualitative (colour, malformations and growing behaviour), and molecular (RAPD and minisatellite) traits. This population consisted of isolates mostly belonging to P. eryngii var. eryngii, var. ferulae or var. nebrodensis, from different regions of Italy. A replicated cultivation trial, with three blocks and three replicates for each strain within the block, was used as an experimental design to calculate trait estimates. Significant differences were observed between strains for basidiome number and weight, while no significant differentiation for quantitative morphological traits was observed between geographical origins and taxonomic groups. Qualitative morphological traits were efficient in differentiating isolates of P. eryngii var. nebrodensis. On average, yield per strain (basidiome weight) was correlated more with basidiome number than with size. The most stable yield traits were basidiome number and weight per strain. An average heritability of 0.31 was estimated for yield related traits. A significant difference between var. ferulae and var. eryngii populations was detected for basidiome production measured as ‘average harvest time’. Molecular markers showed a high level of heterogeneity within populations and a low, but significant, degree of differentiation among populations defined a priori. No population-specific marker was detected and the differential pattern of variation between vars. ferulae and eryngii was due to frequency-dependent alleles. The nebrodensis type was more differentiated from var. eryngii than from var. ferulae using either molecular or qualitative morphological traits.