Genetic variability and protein analysis of three fishes of family Sparidae were studied using Inter-simple sequence repeated (ISSR) markers and Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Species of Sparus aurata, Diplodus sargus and Diplodus cervinus were collected from the Mediterranean Sea in Port Said in Egypt for the present study. In ISSR analysis, twelve ISSR primers (ISSR1, ISSR2, ISSR5, ISSR6, ISSR7, ISSR8, ISSR9, ISSR10, ISSR13, ISSR14, ISSR15 and ISSR20) were used and DNA segments with different lengths were amplified ranging from 180bp with primer ISSR6 to 1000bp with primers ISSR9 and ISSR13. Polymorphism percent was the highest with ISSR-5 (83%) and the lowest with ISSR-20 (20%). Genetic similarity was the highest between Sparus aurata and Diplodus cervinus (73%) and the lowest was between Sparus aurata and Diplodus sargus (67%). SDS-PAGE analysis produced bands ranging from 19 to 200KD and a total of twenty four bands. Twenty three monomorphic bands and one polymorphic band were obtained.