Abstract Nitrogen (N) is quantitatively the main nutrient required by coffee plants, with acquisition mainly by the roots and mostly exported to coffee beans. Nitrate (NO3–) and ammonium (NH4+) are the most important inorganic sources for N uptake. Several N transporters encoded by different gene families mediate the uptake of these compounds. They have an important role in source preference for N uptake in the root system. In this study, we performed a genome-wide analysis, including in silico expression and phylogenetic analyses of AMT1, AMT2, NRT1/PTR, and NRT2 transporters in the recently sequenced Coffea canephora genome. We analyzed the expression of six selected transporters in Coffea arabica roots submitted to N deficiency. N source preference was also analyzed in C. arabica using isotopes. C. canephora N transporters follow the patterns observed for most eudicots, where each member of the AMT and NRT families has a particular role in N mobilization, and where some of these are modulated by N deficiency. Despite the prevalence of putative nitrate transporters in the Coffea genome, ammonium was the preferential inorganic N source for N-starved C. arabica roots. This data provides an important basis for fundamental and applied studies to depict molecular mechanisms involved in N uptake in coffee trees.