Pearl millet is a key food for millions living in semi-arid and arid regions and is a main diet for poorer populations. The genetic diversity existing in the pearl millet germplasm can be used to improve the micronutrient content and grain yield. Effective and organized exploitation of diversity at morphological and DNA levels is the strategy for any crop improvement program. In this study, the genetic diversity of 48 pearl millet genotypes was evaluated for eight morphological traits and eleven biochemical characters. All genotypes were also characterized using twelve SSR and six SRAP markers to evaluate genetic diversity. The significant mean difference between morphological and biochemical traits were detected. The productive tillers per plant varied from 2.65 to 7.60 with a mean of 4.80. The grain yield of genotypes varied more than 3× from 15.85 g (ICMR 07222) to 56.75 g (Nandi 75) with an average of 29.54 g per plant. Higher levels of protein, iron, and zinc contents were found to be present in ICMR 12555 (20.6%), ICMR 08666 (77.38 ppm), and IC 139900 (55.48 ppm), respectively, during the experiment. Substantial variability was observed for grain calcium as it ranged from 100.00 ppm (ICMR 10222) to 256.00 ppm (ICMR 12888). The top eight nutrient-dense genotypes flowered in 34–74 days and had 5.71–9.39 g 1,000 grain weight. Genotype ICMR 08666 was superior for Fe, Zn, K and P. The inter-genotype similarity coefficient at the genetic level, generated using DNA markers, ranged from 0.616 to 0.877 with a mean of 0.743. A combination of morpho-biochemical traits and DNA markers based diversity may help to differentiate the genotypes and diverse genotypes can be used in breeding programs to improve the mineral content in pearl millet.