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Understanding the level of genetic diversity in any population is an important requisite towards strategizing measures for conservation and improvement of stocks. This study focused on the assessment of phylogenetics and molecular divergence of tilapia fish species obtained from two populations (Domita in South-South and Odeda in South-West, Nigeria) using the displacement loop (D-loop) and cytochrome b region of the mitochondrial deoxyribonucleic acid (mtDNA). A total of 28 samples (15 from South-South and 13 from South-West) were used for the genetic analysis. DNA was extracted from the tissue of all the samples using Quik-gDNATM miniPrep kit. The D-loop containing the hypervariable region was sequenced for all samples from the two populations, while cytochrome b (Cyt b) region of mtDNA was only sequenced for samples from South-South population. Chromatograms of the sequences were viewed and edited using Bioedit software. Multiple sequence alignment was carried out using molecular evolutionary genetic analysis (MEGA) software before subsequent genetic analyses. Phylogenetic analysis grouped the samples into two clusters based on population. Also, when the two mitochondrial regions were pooled together, they clustered into two major groups based on mitochondrial regions. Analysis of molecular variance (AMOVA) revealed 37.32% variation within population and 62.68% variation among population with a significant fixation index of 0.627 (p < 0.05). The genetic distance inferred between D-loop regions of South-South and South-West populations was 0.243. Maternal lineage analysis revealed that the origin of tilapia fish from both populations could be traced to Oreochromis spirilus and Oreochromis leucostictus based on mitochondrial D-loop region. The findings of this study revealed molecular divergence among the tilapia populations and may serve as pivot information for the genetic improvement of this important species.