Genetic diversity among three genotypes of tetraploid wheat and four of hexaploid wheat, it was estimated using inter simple sequence repeats (ISSR) and cytogenetic studies. Three genotypes of Triticum dicoccum (Sohag-1, Beni-suef-1 and Beni-suef-3 belonging to Tetraploid wheat and four hexaploid genotypes; Sids-8, Sids-12, Sids-13 and Giza-171 (Triticum aestivum)) sampled from upper and lower Egypt regions; the three durum wheat cultivars and the four hexaploid wheat were assessed. Genetic diversity within wheat genotypes was evaluated using 10 ISSR primers. Of the approximately 431 detected ISSR markers, 117 (27%) were polymorphic with 27 bands per utilized primer pair. Cluster analysis of seven genotypes belonging to the two species by UPGMA cluster analysis based on Jaccard's similarity estimates for ISSR data separated all genotypes into two major clusters depend nearly on their genome makeup. The first one include wheat species possesses AB genomes, while second cluster included wheat genotypes ABD genomes. The genetic similarity coefficients ranged from 0.05 between Beni-suef 3 (Triticum dicoccum) and Sids-8 and Sids-12 of T. aestivum. Concerning chromosome morphology analysis of the three durum wheat genotypes of, the highest value for chromosome length was observed in Sohag-1 (14.84 μm for chromosome 2 A) and the smaller value in Beni-suef 3(5.16 μm for the chromosome 7B). The CI values (centromere index) obtained for durum wheat ranged from 0.48 for 5B to 0.97 for 7A. Thus, according to the CI values for karyotypes of Sohag 1, all chromosomes are metacentric except 5B chromosome. The CI values (centromere index) obtained for durum wheat ranged from 0.50 for 5B to 1.00 for 7A