Macrophomina phaseolina is a soilborne pathogen with a worldwide distribution and a host range of over 500 plant species. To study the genetic diversity and host specialization of this fungus in Iran, plants with signs and symptoms of charcoal rot were collected from 12 provinces during years 2006 to 2008. Following isolation and purification, 52 isolates from 24 host plants were identified through morphological methods and by species-specific primer. Genetic diversity was analyzed using Inter-Simple Sequence Repeat (ISSR) markers. From among the nine tested primers, six sets yielded polymorphic and reproducible bands, used in subsequent analyses. Fingerprint data from all primers were analyzed using UPGMA method and Jaccard´s Coefficient. Cluster analysis divided the isolates into four groups at a 60% similarity level. Group 1 comprised 5.76% of the isolates, including all the hemp from Kashan and Isfahan. Group 2 comprised 68.53% of the isolates, including all the ones from kiwi, soybean, melon, olive, maize, and okra. Golestan isolates from hosts of: maize, sorghum, kiwi, soybean and marigold with 64% similarity being placed together with Qazvin isolates from pumpkin and tomato hosts with 100% similarity may be due to similarity of the host origin. Group 3 being comprised of 13.47% of the isolates, included isolates from Fabaceae and Cucurbitaceae. Most of the Group 3 isolates were collected from arid regions. With isolates of mungbean with 88% of similarity being placed together in this group. Qom and Karaj grown cucumber, Kashan cantaloupe, Qazvin common bean, Khorasan common bean isolates with 75% of similarity were placed together in this third group. Group 4 got comprised of 19.23% of isolates including the ones from Solanaceae, Fabaceae and Cucurbitaceae hosts with no specific correlation to geography. Isolates of cantaloupe from Fars and from Khuzestan with 60% similarity and tomato from Qom and eggplant from Hormozgan with 66% similarity and isolates of sesame from Kerman and Kashan with 90% similarity as well as three isolates of sunflower from Markazi, Fars and Khuzestan with 61% of similarity were placed in this fourth group. The results demonstrate that genetic diversity of M. phaseolina is high among hosts and geographical regions, with host specialization being detectable in some cases.