Background In Europe, Populus nigra generally grows sympatric with the exotic related taxon Populus x canadensis. Natural hybridization followed by introgression and genetic swamping is often mentioned as a potential threat for the conservation of P. nigra. However, as evidence for natural hybridization with P. x canadensis is scarce, the significance of this threat is unclear. Methods After first determining the genotypic diversity within 209 trees of P. nigra collected in western Flanders (northwest Belgium), we assessed natural hybridization events between P. nigra females and P. x canadensis males with diagnostic microsatellite markers. We also studied the possible paternity of the frequently planted P. nigra cv. Italica. Furthermore, we compared the potential seed set of P. x canadensis and P. nigra by counting the number of unfertilized ovules per ovary and the number of ovaries per pistillate inflorescences. Results and conclusions Only two different multilocus microsatellite genotypes were detected within the 209 black poplar trees sampled in western Flanders. In 57 % of the seedlings from the open pollinated black poplar trees, at least one species-specific allele of P. deltoides was detected. P. nigra cv. Italica could not be excluded as a father of one seedling. The potential for seed set and the number of ovules per ovary was significantly higher for P. x canadensis compared to P. nigra. Conclusions The extreme low number of P. nigra genotypes in western Flanders suggests a strong human influence on the species’ genetic diversity. The findings further indicate that natural hybridization events between female P. nigra and male P. x canadensis frequently occur in our study area. Furthermore, P. x canadensis has a higher seed set potential compared to P. nigra. Restoration projects of P. nigra in the valley of the IJzer river should initially aim reforestation measures with a mixture of different black poplar genotypes from neighbouring regions.