Background: In Sub-Saharan Africa, Borassus aethiopum Mart. (African fan palm) is an important non-timber forest product-providing palm that faces multiple anthropogenic threats to its genetic diversity. However, this species is so far under-studied, which prevents its sustainable development as a resource. The present work is a first attempt at characterizing the genetic diversity and population structure of B. aethiopum across nine collection sites spanning the three climatic regions of Benin, West Africa, through the use of microsatellite markers. Results: During a first phase we relied on the reported transferability of primers developed in other palm species. We find that, in disagreement with previously published results, only 22.5% of the markers tested enable amplification of B. aethiopum DNA and polymorphism detection is very low. In a second phase, we generated a B. aethiopum-specific genomic dataset through high-throughput sequencing and used it for the de novo detection of microsatellite loci. Among the primer pairs targeting these, 11 detected polymorphisms and were further used for analyzing genetic diversity. Across the nine sites, expected heterozygosity (He) ranges from 0.263 to 0.451 with an overall average of 0.354, showing a low genetic diversity. Analysis of molecular variance (AMOVA) shows that within-site variation accounts for 53% of the genetic variation. Accordingly, the low number of migrants and positive values of the fixation index (F) in sites from both the Central (Sudano-Guinean) and the Southern (Guinean) climatic regions suggest limited gene flow between sites. The global correlation between genetic and geographic distances is weak; however, our clustering analyses indicate that B. aethiopum palms from Savè (Center) are genetically more similar to those from the North than to samples from other Central sites. Conclusions: In the light of our results, we discuss the use of inter-species transfer vs. de novo development of microsatell