Polyploidy is an important mechanism in angiosperm diversification and evolution. Polyploidization can lead to speciation when the chromosome doubling prevents interbreeding between coexisting cytotypes. In the case of Acacia senegal, different ploidy levels have been reported but the degree and impact of interploidy gene flow in natural populations is not yet resolved. This paper addresses the aspect by characterizing the genetic differentiation among ploidy levels and identifying prezygotic reproductive barriers against interploidy cross-pollination. We genotyped 293 individuals from 10 populations across Senegal using eight microsatellite markers. To study potential pre-zygotic reproductive barriers between diploids and polyploids, we compared the flower morphology and phenology between even-aged diploid and tetraploid trees from a common garden trial that consisted of four different populations originally selected in the natural distribution area of the species in Senegal. The analysis revealed variation among populations in the frequency of polyploids with common co-occurrence of the ploidy levels. The microsatellite markers revealed genetic differentiation between polyploid and diploid trees, and the floral studies identified differences that can act as prezygotic reproductive barriers: tetraploid trees had bigger polyads and stigma cup size compared to diploid trees. Also, polyploid trees on average flowered 9 days earlier than diploids although with overlaps in flowering time. Our results suggest that gene flow among cytotypes of A. senegal is limited and that interploidy pollination is likely to be restricted by differences in flower phenology and morphology.