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Aim: The aims are to study the genetic diversity and population structure of Tilapia fish species in Nigeria using mtDNA D-loop region and time divergence of these various groups of fish to give a baseline information for fish management and conservation programs. Methodology: Blood samples from four species of tilapia were collected with the use of FTA cards for DNA extraction and PCR amplification. A 941bp long fragment, including the D-loop, was subsequently used for analysis. Molecular diversity indices and Tajima’s selective test were determined. A phylogenetic tree was constructed for all the four fish species using UPGMA. The divergence time among the four fish species using MEGA5 software.