This investigation was conducted at the Experimental Farm of Agronomy Department and Genetics Department, Faculty of Agriculture, Minia University during two seasons 2021/22 and 2022/23 to characterize 12 Egyptian bread wheat genotypes by morphological and molecular markers. The relative phenotypic diversity index was higher than 0.60 for all the 19 morphological traits. Cluster analysis classified the 12 genotypes based on 19 traits into 5 clusters. Cluster 1 comprised six lines of 2, 8, 13, 15, 23 and 34 and their two parents of Sids 4 and Giza 168. Cluster 2, 3, 4 and 5 each of composed one genotype; line 4, line 17, line 31 and Giza 171, respectively. The percentages of polymorphism among nine ISSR primers ranged from 20 to 100% with an overall mean of 75.12±10.88%. Ten unique bands were produced by four ISSR primers (HB, HB08, HB10 and 812). These primers were the highest and a more successful in proofing identity of the bread wheat genotypes. It produced (3, 2, 3, and 2 unique bands, respectively) then can be used as a marker to distinguish among them. The dendrogram revealed that the ISSR markers were a successful tool in differentiating amongst the 12 bread wheat genotypes due to their genetic background. Finally, these results showed that both of morphological and ISSR markers could be used as important tools for characterizing the studied Egyptian bread wheat genotypes. It might also provide important information that helps breeders to select the right individuals in plant breeding programs.