Charcoal rot caused by Macrophomina phaseolina is an injurious and economically important disease which causes heavy loss of yield to field and horticultural crops. Thirty-three isolates of M. phaseolina were obtained from different host plants such as: soybean (Glycine max.), melo (Cucumis melo), bean (Phaseolus vulgaris), pea (Pisum sativum), pinus (Pinus sp), blak-eyed susan (Rudbeckia fulgida), kiwifruit (Actiniadia deliciosa), cotton (Gossypium arboreum), and sesame (Sesamum indicum). Pathogenicity test was performed on seeds of melo in in vitro conditions. To study the genetic diversity of isolates PCR-RFLP and RAPD markers were employed. Amplification of the ITS region using ITS5 and LR5 primers produced only one DNA fragment of 1600 bp. Results indicated genetic variability among isolates of M. phaseolina through application of RAPD marker. RAPD data based on five random primers tested on sixteen isolates revealed a high degree of polymorphism in different isolates. UPGMA dendrogram based on RAPD data, produced 9 clusters at the level of 70% similarity. Isolates from the same locations showed a tendency to belong to closer groups, indicating closer genetic relatedness.