The pomegranate (Punica granatum L.), a species of the Lythraceae family, is one of the most significant fruits in Mediterranean and subtropical regions. The key concern for the future of the pomegranate industry is therefore the development of new genotypes of pomegranates that are resistant and yield fruits with increased market demand. In this regard, a new promising pomegranate genotype was selected. This study aims to investigate the use of morphological, chemical, and molecular techniques to characterize a new genotype of pomegranate and evaluate it in comparison to the leading commercial pomegranate varieties in Egypt, Manfalouty and Assiuty. In addition to appreciating the genetic relationship between the new genotype and the two varieties present in Egypt. The studied genotypes displayed marked variability in the weight and shape of the fruits, as well as in the weight of the arils and juice yield. The new genotype (G) recorded the highest values in fruit weight, fruit yield kg/tree, juice percentage, TSS (%) content, total sugars, and anthocyanin in peel and juice. Our results confirm the effectiveness of SCoT and chloroplast cpDNA markers for assessing and identifying pomegranate genotypes. The obtained results showed that the chloroplast cpDNA marker had a relative higher level of polymorphism (72.6%) than that revealed by the SCoT marker (23.82%), and both markers are useful for identification and genetic variability analysis of pomegranate cultivars.