Genetic differentiation and protein analysis of four fish species of family Mugilidae (Mugil cephalus, Mugil capito, Mugil auratus, and Liza seheli), collected from the Mediterranean Sea in Port Said in Egypt, were studied using start codon targeted technique (SCoT) and Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In SCoT technique, eleven SCoT primers (SCoT-1, SCoT-2, SCoT-3, SCoT-4, SCoT-5, SCoT-6, SCoT-21, SCoT-22, SCoT-28, SCoT-35 and SCoT-41) were used and different lengths of amplicons were generated ranging from 170bp with primers SCoT-3 and SCoT-6 to 1600bp with primer SCoT-2. The polymorphism percent ranged from 14% with SCoT-4 primer to 78% with SCoT-28 primer. The genetic similarity was the highest (80%) between Mugil capito and Liza seheli and between Mugil auratus and Liza seheli, and it was the lowest (74%) between Mugil cephalus and Mugil capito and between Mugil cephalus and Mugil auratus. SDS-PAGE analysis generated a total of twenty-four bands with a molecular weight ranging from 19 to 200 KD. Twenty-two monomorphic bands were produced and only two polymorphic bands were generated. Twenty-three bands were obtained from each species.