The main effort of wheat breeder is the detection of genes and to merge them in a particular genotype using most suitable combination. Five Egyptian cultivars of bread wheat (Triticum aestivum L.) were crossed in a half diallel mating design to produce 10 crosses. The genetic potential of embryogenic callus (EC%), plant regeneration (RGP%) response and its association with heading date (HD) and grain yield per plant (GY/P) were investigated. The results showed that GY/P was significantly and positively correlated with EC% and RGP%. The combining ability analysis showed that the magnitudes of general combining ability (GCA) were higher than those of specific combining ability (SCA) for both tissue culture response and agronomic traits. The promising crosses which exhibited desirable SCA effects, showed also high useful heterosis for all studied traits. The magnitudes of additive genetic variance (σ2A) were larger than those of non-additive ones (σ2D) for all studied traits except for number of days to heading. The estimates of narrow sense heritability were 84.56%, 82.13%, 43.46% and 70.28% for the percentage of EC%, RGP%, HD and GY/P, respectively. The genetic similarity percents based on RAPD markers ranged from 76% to 93% between the cultivars. The UPGMA cluster analysis revealed that the cultivars could be divided into two main clusters. The range of Euclidean distances based on morphological characters among the cultivars was relatively wide (4.37–27.87), indicating relatively high amount of phenotypic variation. A significant positive correlation between Euclidean distance and RAPD distance (0.72∗∗) was found.