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The entire collection of cultivated barley germplasm accessions conserved in the Indian National Genebank (INGB) was characterized for nine qualitative and 8 quantitative traits to assess the nature and magnitude of prevailing genetic variability and to develop a core set. A wide range of variability was observed for days to spike emergence (51–139 days), days to physiological maturity (100–152 days), plant height (45.96–171.32 cm), spike length (3.44–13.73 cm), grain number/spike (10.48–82.35), and 100-grain weight (1.20–6.86 g). Initially, seven independent core sets were derived using 3 core construction tools– MSTRAT, PowerCore, and Core Hunter 3 by employing the maximization method, heuristic sampling, and optimisation of average genetic distances, respectively. The core set-3 generated by Core Hunter 3 by simultaneous optimisation of diversity and representativeness, captured maximum genetic diversity of the whole collection as evident from the desirable genetic distance, variance difference percentage (VD; 87.5%), coincidence rate of range (CR; 94.27%) and variable rate of coefficient of variance (VR; 113.8%), which were more than threshold value of VD (80%), CR (80%), and VR (100%) required for good core collection. The coefficient of variation and Shannon–Weaver diversity indices were increased in the core set as compared with the whole collection. The low value of Kullback-Leibler distance (0.024–0.071) for all traits and quantile-quantile plots revealed a negligible difference between trait distribution patterns among the core set and entire assembly. Correlogram revealed that trait associations and their magnitude were conserved for most of the traits after sampling of the core set. The extraction of the INGB barley core set and identification of promising accessions for agronomically important traits in different genetic backgrounds will pave the way for expedited access to genetically diverse and agronomically important germplasm for barley breeding.