Accurate characterization of crops or fruits leads to successful improvements in breeding programs. Mango (Mangifera indica L.) is the second most common fruit in Egypt. It is a heterozygous plant; consequently, the possibility of incorrect characterization is significant. Therefore, this study used six accessions of two M. indica cultivars, ‘Keitt’ and ‘Ewais’, to produce precise molecular characterization and prove their genetic relatedness using inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) markers. Pollen morphology was studied for the first time for these two Egyptian cultivars to determine their correlation. Morphological traits were quantitatively and qualitatively investigated. In addition, phytochemical analyses were performed to correlate the six mango accessions. One of these accessions (accession 3) was commercially identified as ‘Ewais’ cv., but the obtained result revealed that it does not belong to ‘Ewais’ cv. This finding was proved molecularly using ISSR and SCoT markers and morphologically. Similarly, phytochemical analyses revealed a difference between accession 3 and the other accessions under investigation. Polymorphic information content (PIC) and resolving power (RP) data revealed that the SCoT technique was more informative than ISSR for studying these genotypes of mango. Accordingly, the present findings prove the necessity for molecular, morphological, palynological, and biochemical investigations for accurate characterization of mango genotypes and genetic relatedness for improving breeding programs.