Genetic diversity of species is an important baseline for the domestication process. In Cameroon, Prunus africana, an important and threatened medicinal tree, is among the priority species for domestication. The bark extract has been used to treat various diseases; mainly benign prostatic hyperplasia which affects men above the age of 50. As little is known about the genetic diversity of P. africana in Cameroon, we aimed to determine the genetic diversity and differentiation of several P. africana populations in the western provinces, using sets of chloroplast DNA markers and nuclear microsatellites previously developed for Prunus species. Genetic diversity in the observed populations was considerable and genetic differentiation between populations proved substantial with 21% of the total observed variation detected among populations, revealing a distinct genetic structure among certain populations. However, the lack of correlation between genetic and geographic distances does not support isolation by distance (IBD). The analysis of chloroplast DNA haplotypes revealed no strong phylogeographic component in the genetic structure observed in the western populations of P. africana in Cameroon. The outcome of this study will contribute to improve the genetic characterization of P. africana for its better domestication and conservation in the Cameroon agroforestry system.