The mountain ecosystem of the Central Himalayan Region is known for its diversity of crops and their wild relatives. In spite of adverse climatic conditions, this region is endowed with a rich diversity of millets. Hence, the aim of the present study was to explore, collect, conserve and evaluate the diversity of barnyard millet (Echinochloa frumentacea) to find out the extent of diversity available in different traits and the traits responsible for abiotic stress tolerance, and to identify trait-specific accessions for crop improvement and also for the cultivation of millets in the region as well as in other similar agro-ecological regions. A total of 178 accessions were collected and evaluated for a range of morpho-physiological and biochemical traits. Significant variability was noted in days to 50% flowering, days to 80% maturity, 1000 seed weight and yield potential of the germplasm. These traits are considered to be crucial for tailoring new varieties for different agro-climatic conditions. Variations in biochemical traits such as lipid peroxidation (0·552–7·421 nmol malondialdehyde formed/mg protein/h), total glutathione (105·270–423·630 mmol/g fresh weight) and total ascorbate (4·980–9·880 mmol/g fresh weight) content indicate the potential of collected germplasm for abiotic stress tolerance. Principal component analysis also indicated that yield, superoxide dismutase activity, plant height, days to 50% flowering, catalase activity and glutathione content are suitable traits for screening large populations of millet and selection of suitable germplasm for crop improvement and cultivation. Trait-specific accessions identified in the present study could be useful in crop improvement programmes, climate-resilient agriculture and improving food security in areas with limited resources.