This study aimed at assessing the genotype variation among six tuna fish collected from certain Libyan and Egyptian coastal regions and the molecular characterization due to the prevailing ecological parameters using SRAP and ISSRs. In addition, the current work addressed the impact of some ecological factors in water for tuna biodiversity analysis. ISSR analysis is useful for the assessment of genetic diversity among the genotypes of bluefin tuna. It is possible to conclude that, the short the distance between any two locations, the more they increase the similarity between the two genotypes collected from these two locations. SRAP analysis is useful for assessing the genetic diversity among the genotypes of bluefin tuna. The SRAP results confirmed that when the distance between the locations increases, the tuna genotypes become distantly related and vice versa. The ISSR and SRAP combined data revealed that the highest similarity value among these six tuna genotypes (0.90) was recorded between ELkhoms and Serit genotypes (the two most closely related genotypes). On the other hand, the lowest value (0.60) was recorded between the Egyptian genotype and the Sebrata genotype and between the Egyptian genotype and ELkhoms genotype, with the same similarity value (the two most distantly related genotypes). It is possible to conclude that, the similarity value increased as much as distances between locations decreased and vice versa. The ecological impact of the four measured ecological factors revealed that the variation in these factors could act as barriers that decrease the tuna fish migration and thus decrease the chances of mating between the different genotypes among the different locations. This low chance of mating between the tuna fish genotypes could lead to the speciation proved by means of molecular investigation.