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The present study was aimed to evaluate the genetic variability among three fish species of genus Puntius, Puntius semifasciolatus, Puntius conchonius and Puntius oligolepis (family: Cyprinidae) using various genetic markers (SDS-Page and ISSR). The electrophoretic analysis of protein was revealed 29 bands of molecular weight ranging from 11 to 132KD with polymorphism 28%. A total of eleven ISSR primers produced 123 total bands their size ranged from 79 to 1185bp and the percentage of polymorphism ranged from 91% to 100%. Results analysis showed that the percentages of genetic relationship among three species were 33% and 47%. The results of the study revealed that Puntius semifasciolatus and Puntius conchonius have higher genetic similarity 47% while Puntius semifasciolatus and Puntius oligolepis exhibited lower genetic similarity 33%. Considering all the gained data, it is evident that molecular detection of SDS-Page and ISSR are suitable tools in assessing genetic variation among fish species. These markers provide interesting tools for breeding new varieties of Egyptian fish.