• Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. Here, we report on the characterization of the ammonium transporter (AMT) family in the perennial species Populus trichocarpa. • In silico analysis and expression analysis of AMT genes from poplar was performed. In addition, AMT1;2 and AMT1;6 function was studied in detail by heterologous expression in yeast. • The P. trichocarpa genome contains 14 putative AMTs, which is more than twice the number of AMTs in Arabidopsis. In roots, the high-affinity AMT1;2 strongly increased upon mycorrhiza formation and might be partly responsible for the high-affinity ammonium uptake component measured in poplar. Transcript level for the high-affinity AMT1;6 was strongly affected by the diurnal cycle. AMT3;1 was exclusively expressed in senescing poplar leaves. Remarkably AMT2;1 was highly expressed in leaves while AMT2;2 was mostly expressed in petioles. Specific expression of AMT1;5 in stamen and of AMT1;6 in female flower indicate that they have key functions in reproductive organ development in poplar. • The present study provides basic genomic and transcriptomic information for the poplar AMT family and will pave the way for deciphering the precise role of AMTs in poplar physiology.