This research was conducted to study the nature and magnitude of gene action and combining ability effects for different traits in sesame in order to identify parents and hybrids for future breeding programs. Nine sesame genotypes including five Iranian breeding lines and four Asian genotypes were crossed in a complete diallel mating design. The parents, 81 F 1 s, and 45 F 2 s were grown in a randomized compelled block design with three replications during 2006-2008 in Agricultural farm, Isfahan University of Thechnology. Analysis of variances indicated highly significant differences among the genotypes. Variances due to GCA and SCA were significant for all the traits in both F1 and F2 generations. The results showed that additive gene actions had greater effects on inheritance of days to flowering and maturity, plant height, high to first fruiting node, number of branches/plant, 1000 seed weight, and oleic acid content. However, number of seeds/capsules, seed yield, oil content and the percentage of linoleic acid were largely controlled by dominance genetic effects, Whereas both additive and non-additive gene actions were important in genetic control of capsules/plant, seed protein, stearic and palmitic acid content. The results of generation mean analysis and jont scaling test showed that additive-dominant model had essential adequacy in description of variation in all traits studies in cross of Varamin2822 and Indian lines. The best combiners included EM for plant height and height to first fruiting node, Varamin2822 and Yekta for seed yield, capsules per plant and seeds per capsule, D1 for seed oil content, Indian for oleic and Yekta for linoleic and stearic acid contents. A considerable heterosis was also observed for the studied traits in some cross combinations and the highest heterobeltiosis values of 62.2 and 85% were obtained for capsules/plant and seed yield, respectively. The overall results indicated that crosses EM×Iraq22 and EM×Varamin2822 could be promising for development of high oil yielding hybrids. The cross Varamin2822×D1 could be utilized for the selection of high yielding genotypes in advance segregating generations. To quantify genetic diversity among 24 sesame genotypes based on agro-morphological and ISSR marker traits, cluster analysis was performed and grouped the genotypes into five separate clusters according to agro-morphological data. The 13 ISSR primers chosen for molecular analysis revealed 170 bands, of which 130 (76.47%) were polymorphic. The generated dendrogram based on ISSR profiles divided the genotypes into seven groups. These two marker systems reflected different aspects of genetic variation among the genotypes. So, the complementary application of both types of information is recommended to maximize the efficiency of