Abstract Context: Population-specific attributes typically determine the behavior of human genotypes and traits in individuals. Examining the genetic relationships between populations presents an opportunity to observe patterns of genetic changes over time. By comparing populations, it is possible to see factors that may have sparked the evolution of a trait or genotype as well as the genetic diversity of the populations. Genetic diversity in the groups in Nigeria is still largely undefined. This can be achieved with the use of genetic markers. Aims: To determine the genetic relationships between Igbo, Ibibio, Yoruba, Tiv, and Hausa ethnic groups using nine short tandem repeat DNA markers. Settings and Design: Observational studies of 250 consenting participants. Methods and Material: Individuals of Igbo, Yoruba, Hausa, Ibibio, and Tiv were randomly sampled from their ethnic homes in Nigeria at participating institutions. DNA was extracted from blood samples using commercial DNA kits, and tested for nine autosomal short tandem repeat loci alleles using PCR and electrophoresis. The allele types and sizes were read and recorded for each locus for every individual. Statistical Analysis Used: Allelic frequencies, population pairwise genetic distances (FST and RST, analysis of molecular variance (AMOVA), and principal component analysis (PCA) were calculated using GenAlEx v6.502. Results: The fixation index (FST) was between 0.001 and 0.500 in the five populations. The estimated variation was 99.98% within individuals and 0.10% among populations. Four heterogeneous clusters were observed with three axes accounting for 32.86% variation. Conclusions: The genetic relationships closely mirrored linguistic groupings showing that language may have shaped early interaction and ultimately gene transfer between these groups.