Fusarium solani f.sp. cucurbitae (F.s.c.) Snyder and Hansen is one of the most important fungi causing serious damages to the cucurbits production areas in Tunisia. The goal of this investigation was to study the genetic characterization of F. solani f.sp. cucurbitae Tunisian population. Ten isolates of F.s.c. (F.s.c.1 to F.s.c.10) collected from infected watermelon plants in numerous regions of Tunisia; were subjected to PCR identification using ITS1-ITS4 primers pair, and Inter-simple sequence repeat (ISSR) technique. Results obtained confirmed the presence of F. solani f.sp. cucurbitae race 1 in Tunisia. ISSR dendrogram of the ten F.s.c. isolates generated by bi- and trinucleotide primers; and conducted by the unweight pair grouping using mathematical averaging (UPGMA) method from Nei's genetic distance, revealed the presence of similarities between the isolates that ranged from 31 to 84%. The isolates F.s.c.8 (collected from Gabes) and F.s.c.4 (collected from Sidi Bouzid), were genetically dissimilar presenting the lowest similarity coefficient. In contrast; isolates F.s.c.3 (collected from Sfax) and F.s.c.1 (collected from Beja) were similar. Cluster analysis based on UPGMA of the ISSR markers data assembled F.s.c. isolates into two major groups. These results are helpful to develop integrated management and future breeding programs for plant resistance.