We investigated the origin and diversification of the high-affinity nitrate transporter NRT2 in fungi and other eukaryotes using Bayesian and maximum parsimony methods. To assess the higher-level relationships and origins of NRT2 in eukaryotes, we analyzed 200 amino acid sequences from the Nitrate/Nitrite Porter (NNP) Family (to which NRT2 belongs), including 55 fungal, 41 viridiplantae (green plants), 11 heterokonts (stramenopiles), and 87 bacterial sequences. To assess evolution of NRT2 within fungi and other eukaryotes, we analyzed 116 amino acid sequences of NRT2 from 58 fungi, 40 viridiplantae (green plants), 1 rhodophyte, and 5 heterokonts, rooted with 12 bacterial sequences. Our results support a single origin of eukaryotic NRT2 from 1 of several clades of mostly proteobacterial NNP transporters. The phylogeny of bacterial NNP transporters does not directly correspond with bacterial taxonomy, apparently due to ancient duplications and/or horizontal gene transfer events. The distribution of NRT2 in the eukaryotes is patchy, but the NRT2 phylogeny nonetheless supports the monophyly of major groups such as viridiplantae, flowering plants, monocots, and eudicots, as well as fungi, ascomycetes, basidiomycetes, and agaric mushrooms. At least 1 secondary origin of eukaryotic NRT2 via horizontal transfer to the fungi is suggested, possibly from a heterokont donor. Our analyses also suggest that there has been a horizontal transfer of nrt2 from a basidiomycete fungus to an ascomycete fungus and reveal a duplication of nrt2 in the ectomycorrhizal mushroom genus, Hebeloma.