Natural hybridization has long been recognized as a means for gene flow between species and has important evolutionary consequences. Although hybridization is generally considered to be symmetrical, with both hybridizing species being equally likely to be the male or female parent, several studies have demonstrated the presence of asymmetrical hybridization and introgression from one species to the other. We investigated the direction of natural hybridization between two sympatric forest tree species in North America (Populus deltoides  Bartr. ex Marsh. and Populus balsamifera L.) using species-specific single nucleotide polymorphism (SNP) markers in both the nuclear and chloroplast genomes. All natural hybrid individuals, identified from morphological traits, had nuclear alleles corresponding to both parental species, while the chloroplast genotypes showed similarity to P. deltoides, indicating asymmetrical hybridization with P. deltoides as the maternal and P. balsamifera as the paternal donor species. This observed asymmetrical hybridization may be attributable to cytonuclear interactions.