Evaluation of genetic diversity within germplasm collections and identification of trait-specific germplasm is a basic requirement for plant breeders. A total of 221 Indian pearl millet collections from the National Genebank were characterised and evaluated for 27 agro-morphological descriptors. Considerable variation was observed for all characters. Frequency distribution analysis showed predominance of cylindrical and compact spike, grey seeds, earliness (less than 40 days to spike emergence). Hierarchical clustering method was used for classifying 221 pearl millet accessions based on agronomic and disease resistance traits, which resulted into three clusters. Clusters 1, 2 and 3 comprised 91, 54 and 76 accessions respectively. There was high correspondence between the geographic collection sites of accessions and their inclusion in particular clusters. In addition, principal component analysis was used for data reduction and generating biplot. First four principal components explained 66.43% of total variability. Among the traits analysed, plant height, nodes/plant, days to spike emergence, number of tillers, leaf width and leaf length are major contributor towards phenotypic diversity. Further the trait-specific germplasm were identified for agronomic traits, disease resistance, popping and antioxidants activity, namely for earliness (IC343664, IC343689, IC343661, IC309064), spike girth (IC283693, IC283842, IC367638), dual purpose with high grain and fodder yield (IC283705, IC283745, IC283885 and IC335901 and so on). Four accessions of pearl millet germplasm viz., IC309064, IC393365, IC306465 and IC283866, were observed as multiple disease resistant. This study suggested that application of appropriate techniques and their interpretations provide more efficient way to identify potential accessions and improve the utilisation of germplasm collections in plant breeding.