This study investigated the transcriptomic mechanisms underlying melatonin accumulation and the enhancement of salt tolerance in hull-less barley seeds subjected to zinc sulphate stress. Following zinc sulphate treatment, hull-less barley seeds demonstrated increased melatonin accumulation and improved salt tolerance. Through transcriptome analysis, the study compared gene expression alterations in seeds (using the first letter of seed, this group is marked as 'S'), seeds treated with pure water (as the control group, is marked as 'C'), and germinated seeds exposed to varying concentrations of zinc sulphate (0.2 mM and 0.8 mM, the first letter of zinc sulphate, 'Z', is used to mark groups 'Z1' and 'Z2'). The analysis revealed that 8176, 759, and 622 differentially expressed genes (DEGs) were identified in the three comparison groups S.vs.C, C.vs.Z1, and C.vs.Z2, respectively. Most of the DEGs were closely associated with biological processes, including oxidative-stress response, secondary metabolite biosynthesis, and plant hormone signaling. Notably, zinc sulphate stress influenced the expression levels of Tryptophan decarboxylase 1 (), Acetylserotonin O-methyltransferase 1 (), and Serotonin N-acetyltransferase 2 (), which are key genes involved in melatonin synthesis. Furthermore, the expression changes of genes such as Probable WRKY transcription factor 75 () and Ethylene-responsive transcription factor ERF13 () exhibited a strong correlation with fluctuations in melatonin content. These findings contribute to our understanding of the mechanisms underlying melatonin enrichment in response to zinc sulphate stress.