The development of next‐generation sequencing technologies allows researchers to address complex problems in species delimitation, especially for non‐model organisms. The taxonomic status of North American Nyssa species has long been debated and remains controversial. To elucidate the genetic structure and phylogenetic relationships of the five currently recognized North American Nyssa species, we conducted whole‐genome sequencing of representative individuals and identified genome‐wide single‐nucleotide polymorphisms (SNPs) by utilizing the recently released chromosome‐level assembly of Nyssa sinensis genome. Population genetic and phylogenetic analyses consistently inferred four well‐supported genetic clusters from our sampled individuals, that is, N. aquatica, N. ogeche, N. sylvatica, and N. biflora–N. ursina. Although the identification of N. biflora and N. ursina is primarily based on the morphological characteristics of leaves and drupes, the present evidence, including our principal components analysis of leaf morphological traits, strongly supports the taxonomic designation of N. biflora and N. ursina as a single species. In addition, these four genetic clusters were grouped into two major clades, that is, clade 1 (N. aquatica and N. ogeche) and clade 2 (N. sylvatica and N. biflora–N. ursina). Despite the fact that no evidence of widespread gene flow was found between these two major clades, our analyses revealed the possibility of introgression from N. sylvatica into N. biflora, albeit at a relatively low frequency. This study demonstrates the use of whole‐genome sequences as a promising avenue for delimiting species boundaries and further advocates for an integrative approach in the assessment of species delimitation. We conducted whole‐genome sequencing of representative individuals for five currently recognized North American Nyssa species. Population genetic and phylogenetic analyses of genome‐wide SNPs consistently inferred four well‐supported genetic clusters, that is, Nyssa aquatica, N. ogeche, N. sylvatica, and N. biflora–N. ursina, and the recognition of N. biflora and N. ursina as a single species was also supported by PCA of leaf morphological characters. These four genetic clusters were further grouped into two major clades, that is, clade 1 (N. aquatica and N. ogeche) and clade 2 (N. sylvatica and N. biflora–N. ursina), and no evidence of widespread gene flow was found between the two major clades.