Populus, a deciduous tree species of major economic and ecological value, grows across the range in which trees are distributed in the Northern Hemisphere. Patterns of DNA variation are often used to identify the evolutionary forces shaping the genotypes of distinctive species lineages. Sodium/hydrogen (Na+/H+) antiporter genes have been shown to play a central role in plant salt tolerance. Here, we analyzed DNA nucleotide polymorphisms in the Na+/H+ antiporter (NHX and SOS1) gene families across 30 different Populus species using several methods of phylogenetic analysis and functional verification. NHX and SOS1 gene families in the genus Populus have expanded from the state in their common ancestors by duplication events, and their distinct lineages have been retained. Signals of positive selection at certain amino acid sites in different members of the Na/H antiporter gene families show that the dynamics that drive the evolution of each gene vary. SOS1 has undergone duplication in Populus euphratica and been subjected to adaptive evolution in section Turanga; the paralog of PeSOS1 (PeSOS1.2) can complement the Escherichia coli mutant EP432; and the expression pattern of PeSOS1.2 is different from that of PeSOS1, a fact which may have been beneficial for P. euphratica, conferring a fitness advantage in saline habitats. The divergent evolution of the individual members of the NHX and SOS1 gene families is likely to have been influenced by the varied ecological and environmental niches occupied by the different poplar species, giving rise to evolutionary footprints that reflect the specific functions and subcellular localizations of the proteins encoded by these genes.