Eighty‐six Heterobasidion annosum isolates, mainly belonging to the F intersterility group and obtained from 32 different geographical localities in Italy, were subjected to genetic analysis by the Random Amplified Polymorphic DNA (RAPD) markers. The similarity between F and S groups was higher than that between F and P. In UPGMA Cluster Analysis, the F isolates originating from the same locality usually grouped in the same cluster. The isolates also showed a tendency to group at the level of larger geographical areas. Within the F group, isolates from the south of the Italian peninsula showed the highest genetic variation and northern isolates from the Alpine regions showed the lowest. This indicates a gradual cline along the peninsula. The genetic variability in the Italian F group is discussed in relation to the past and present distribution of the host species in Italy and Europe.