Graminaceous plants take up iron (Fe) from soil using specialized chelating agents known as phytosiderophores, which largely comprise mugineic acids (MAs). Biosynthesis of MAs involves three enzymes, of which nicotianamine aminotransferase (NAAT) catalyses the key step in the synthesis of 2’-deoxymugineic acids (DMA, the precursors of MAs). In the present study, a total of 24 TaNAAT genes distributed on 15 of the 21 bread wheat chromosomes were identified using the whole genome sequence (as against six genes reported in two earlier reports; rice genome is also known to have only six NAAT genes). We also identified NAAT genes in diploid and tetraploid relatives of bread wheat. Two gene duplication events involving NAAT genes were also identified, one in Triticum urartu (donor of the A sub-genome) and the other in Aegilops tauschii (donor of the D sub-genome). In the promoter regions, a number of cis-regulatory elements were also identified for responses to biotic and abiotic stresses and to different developmental stages. Phylogenetic analysis using NAAT proteins of wheat and seven other plant species (including T. urartu, Ae. tauschii, T. turgidum, Oryza sativa, Zea mays, Hordeum vulgare, and Brachypodium distachyon) led to the identification of six clusters. Both in silico and qRT-PCR expression analyses indicated relatively higher expression of TaNAAT genes in shoot and root of genotypes with low Fe content. The results provided insights into the structure and function of TaNAAT genes in wheat, which may further help in planning strategies to develop high yielding wheat varieties tolerant to Fe deficiency.