A long history of hybridization and introgression in the evolution of Betula has made the use of chloroplast genes to infer interspecific relationships prone to errors. Single and low copy nuclear genes, however, may offer some hope for accurately reconstructing phylogenetic relationships of Betula and inferring the origin of polyploid species. In this study, sequences of the third intron of nuclear nitrate reductase (Nia) were used to estimate relationships of known diploid species of Betula. Two distinct types of the Nia gene were observed in Betula. Sequences of one type were gathered from all sampled species; those of the second type, obtained from only B. nana, were not used in phylogenetic analyses. Cloned sequences of the Nia gene varied little within individuals or species. Maximum parsimony and likelihood analyses generated robust phylogenies with similar relationships. Betula alnoides and B. maximowicziana of subgenus Betulaster formed a well supported clade, which is characterized by the racemose pistillate inflorescence. Shrubby species (B. nana and B. michauxii) of a traditionally recognized group did not form a clade. Instead, B. nana was most closely related to white-barked species (e.g., B. pendula, B. platyphylla, and B. populifolia) and together they formed a clade with B. nigra. In contrast, B. michauxii is closely allied with B. lenta. Our results suggest that subgenera Betulaster and Betula are monophyletic, while subgenera Chamaebetula and Neurobetula are polyphyletic.