Iron deficiency causes chlorosis in many plant species, resulting in yield loss and poor quality. Many tree species including poplar are susceptible to iron deficiency. Trees suffering from iron deficiency often show interveinal chlorotic leaves and in severe cases, branches or an entire tree may die. In this study, two trees of Populus tremula L.‘Erecta’ growing near each other but with contrasting leaf color phenotypes were used to study the causes of chlorosis and the mechanisms of tolerance or susceptibility to iron chlorosis in poplar. A leaf analysis revealed that the iron deficiency tolerant tree (PtG) had a higher level of dry matter content, chlorophyll (a+b), Chl a/b ratio, Zn and Fe content than the iron chlorosis susceptible tree (PtY). A hydroponic culture confirmed the differences in aforementioned physiological parameters between PtG and PtY responding to iron deficiency. Two iron-regulated transporter genes (PtIRT1 and PtIRT3), the native promoter of the PtIRT1 gene (PtIRT1-pro), and two basic helix-loop-helix (bHLH) transcription factors (PtFIT and PtIRO) were cloned and characterized for their responses to iron deficiency in PtG and PtY. Deduced amino acid analysis revealed that PtIRT1, PtIRT3, PtFIT, and PtIRO in PtG were identical to those in PtY. Phylogenetic and putative domain analyses showed that PtIRT1, PtFIT, and PtIRO may function in iron homeostasis, while PtIRT3 may play a role in zinc transport in poplar. The expression of PtIRT1 and PtFIT are root-specific and up-regulated by iron deficiency. The expression of a GUS gene derived by PtIRT1-pro in tobacco was also up-regulated by iron deficiency, but was not root-specific. The expression of PtIRT3 is ubiquitous and up-regulated by iron deficiency, but significantly down-regulated by zinc deficiency. A high correlation in the expression between PtFIT and PtIRT1 was observed in PtG, but not in PtY. Transgenic poplars overexpressing PtIRT1 or PtIRT3 did not have enhanced Fe accumulation; however, an enhanced tolerance to iron deficiency was found in transgenic plants overexpressing PtFIT. The results suggested that the transcription factor PtFIT may be involved in iron deficiency response through regulation of PtIRT1 and PtFIT itself may be regulated by other factors in poplar.