Abstract: Microsatellite markers MCW88, MCW150, ADL171 and ADL206 were used to analyze genetic variability and divergence of chicken populations of different geographical location in Ogun and Ondo States, Nigeria. The chicken populations included 16 Frizzle feathered (Ff), 16 Normal feathered (Nf), 16 Naked neck (Nn), 16 Bovan nera (Bn) and 16 Black harco (Bh) making a total of 80 isolated genomic DNAs. Sample of DNAs were amplified by polymerase chain reaction (PCR) and separated on 1% agarose gel. 84 alleles were detected by the selected markers. The number of identified alleles per locus ranged from 2 to 14. High value of FST (0.1167) across loci revealed a substantial degree of population differentiation. In the majority of the chicken populations high heterozygosity and some level of inbreeding were determined. The heterozygosity ranged from 0.5315±0.08 to 0.6989±0.03 while inbreeding ranged from 0.2168±0.19 to 0.4448±0.09. The Nei’s chord genetic distance (Da) was also calculated using Microsatellite Analyzer. Dendrogram developed using neighbor-joining method showed that Bovan nera(Bn) vs. Black harco(Bh) formed the first group (0.1404), followed by Frizzle feathered (Ff) vs. Bovan nera(Bn) (0.1825), Frizzle feathered(Ff) vs. Normal feathered(Nf) (0.2255), Normal feathered(Nf) vs. Naked neck(Nn) (0.3699), respectively. These results highlight the applicability of microsatellite markers to determine rare allele, inbreeding coefficient and genetic divergence in five chicken populations in Ogun and Ondo States, Nigeria. It was concluded that the markers were useful in revealing genetic information present in the five chicken populations.