Nitrogen (N) is an essential macronutrient for the growth and development of plants, but excessive use of nitrogen fertiliser in agriculture can result in environmental pollution. As a preferred nitrogen form, ammonium (NH ₄ ⁺) is absorbed from the soil by the plants through ammonium transporters (AMTs). Therefore, it is important to explore AMTs to improve the efficiency of plant N utilisation. Here, we performed a comprehensive genome-wide analysis to identify and characterise the AMT genes in barley ( HvAMTs), which is a very important cereal crop. A total of seven AMT genes were identified in barley and further divided into two subfamilies ( AMT1 and AMT2) based on phylogenetic analysis. All HvAMT genes were distributed on five chromosomes with only one tandem duplication. HvAMTs might play an important role in plant growth, development, and various stress responses, as indicated by cis- regulatory elements, miRNAs, and protein interaction analysis. Further, we analysed the expression pattern of HvAMTs in various developmental plant tissues, which indicated that AMT1 subfamily members might play a major role in the uptake of NH ₄ ⁺ from the soil through the roots in barley. Altogether, these findings might be helpful to improve the barley crop with improved nitrogen use efficiency, which is not only of great significance to the crop but also for land and water as it will reduce N fertiliser pollution in the surrounding ecosystem.