Sampling was carried out from different fields in Hamedan province, Iran during the years 2012 and 2013. Among the 300 obtained isolates of Alternaria spp. after morphological identification, it was revealed that A. alternata isolates from potato had the highest frequency distribution. Due to the abundance of this species in this region which is considered as a major potato producing province in the country, genetic diversity of its isolates were assessed using ISSR markers. Among A. alternata isolates, 11 isolates screneded from nine different regions on four potato cultivars were selected. A total of 15 ISSR primers of UBC group were used to investigate the genetic diversity of these isolates. Out of 15 primers used in this study, 5 primers had favorable results and produced a significant number of bands. Based on the results of cluster analysis using the Jaccard's coefficient, isolates were divided into two main groups and there was some correlation between the grouping of isolates regarding their geographic location, pathogenicity and potato cultivars.