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Evaluation of genetic diversity in medicinal plants is one of the most important evolutionary and breeding goals. Recent developments in polymerase chain reaction brings the possibility of evaluation the individuals of a population in more sites of genome, and among different DNA molecular markers, the ISSR marker was successfully used in study of the genetic diversity of different plants. The genetic diversity of 43 individuals from five populations of Styrian Pumpkin (Cucurbita pepo var. styriaca) and 4 individuals from one population of C. maxima which cultivated in Shahid Beheshti University collection (Tehran, Iran) was investigated using 12 ISSR markers. Totally, 83 scorable bands were produced by ISSR markers and the mean for the produced band for each marker was 6.91 and 100% of scorable bands were polymorphic. Dendrogram was illustrated based on Jaccard coefficient similarity matric and algorithm of Complete Linkage. Based on cluster analysis, individuals of populations divided into five main groups. The results of the grouping through principal coordinate analysis and cluster analysis showed that groupings by the two mentioned methods were consonant with each other and have made a similarity grouping. The cophenetic coefficient was calculated as 0.97. Totally, the results of present study showed that the some of used ISSR markers could be useful for future study of genetic variation in Cucurbita pepo.