The crucial involvement of tubby like proteins (TLP) in the response to defense signaling through plant hormones under different stresses is well established. However, there is currently no comprehensive analysis of HvTLP genes in barley. A comprehensive investigation of TLP family in barley was carried out. This included the identification and characterization of genes, phylogenetic analysis, assessment of gene structures and motifs, prediction of cis-elements, and examination of gene expression under various stress conditions. Two barley cultivars, Nosrat (stress tolerant) and Nimroz (stress susceptible), were also studied for physiological traits under abiotic stress conditions. The results indicated that 10 HvTLP genes were highly expressed in leaf tissues at 24 and 48 h after stress. The phylogenetic analysis of HvTLP protein sequences in barley revealed three distinct clusters. Notably, the promoter regions of HvTLP3 and HvTLP7 had the highest number of cis-elements, suggesting their role in responding to abiotic stresses. These genes also contained STRE, MYB, and MYC elements in their promoter regions, which are known to play a significant role in stress response. Additionally, the expression of HvTLP genes was significantly higher in Nosrat cultivar as compared to Nimroz cultivar under abiotic stresses. This was consistent with the elevated levels of chlorophyll a/b, total chlorophyll, flavonoids, carotenoids, and anthocyanin in the Nosrat cultivar compared to the Nimroz cultivar in response to stress. These findings highlight the potential of HvTLP genes as targets for genetic manipulation to develop stress-tolerant crop plants.