Understanding the genetic diversity of cowpea (Vigna unguiculata L. Walp.) landraces is useful for effective characterization and ex-situ conservation of germplasm. The analysis of genetic diversity of eighteen cowpea landraces collected from five agro-ecological zones in Nigeria was reported in this study. Five individuals per landrace were genotyped with six polymorphic microsatellite markers. Three to 5 alleles with a mean of 3.833 were detected. Mean Polymorphic information content (PIC) and observed heterozygosity of the markers were 0.5721 and 0.2433, respectively. Analysis of Molecular Variance (AMOVA) showed that variation due to agroecological zone constituted 24%, while variations among and within landraces as well as within individuals constituted 25%, 17% and 33%, respectively. Landraces collected from the humid rainforest zone showed high within landrace diversity and were not significantly different (P ≥ 0.001) from other landraces collected from the same zone. Landraces from the savannah zones showed low within landrace diversity and homozygous across all loci. Consequently, among landrace diversity was higher in the savannah zone with landraces collected from guinea savannah been the most diverse, followed by landraces from the derived savannah and Sudan savannah. Mantel test showed positive and significant correlation (r= 0.377, p= 0.01) between genetic and geographical distance of landrace collections. The findings are important for up-to-date characterizations of cowpea germplasm in Nigeria for improved breeding programs. Keywords: Genetic diversity, cowpea, Alleles, Polymorphic information content, AMOVA,
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