Hybridization frequently occurs among poplars, both naturally and artificially, hindering identification. Over 32 million clonal poplars, predominantly hybrids, have been planted throughout the Canadian prairies over the past century, making confirmation of genomic identity important. We developed a genotyping assay that rapidly diagnoses four compatible Populus species ( Populus balsamifera L. and Populus deltoides Bartr. ex Marsh.: indigenous, Populus laurifolia Ledeb. and Populus nigra L.: exotics) and their hybrids found throughout this ecozone. First, we sequenced 23 genes from representative provenances of the four Populus species to discover single nucleotide polymorphisms (SNPs). Second, we developed and validated a medium-throughput genotyping assay of 26 diagnostic SNPs within these genes. We used this assay to genotype 198 trees from natural populations as well as 30 clones (pure species and hybrids), including those broadly distributed by Agriculture and Agri-Food Canada’s Agroforestry Development Centre since 1910. This suite of SNPs has the resolving power to correctly identify pure species and hybrids of Populus. We confirmed the identity of clones of well-documented origin, complex hybrids with exotic components, and paternity of open-pollinated progenies from breeding programs. This diagnostic tool should prove useful for efficient molecular fingerprinting of breeding material and for further studies of interspecific gene flow on the Canadian prairies.