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The possibility of developing deep-sowing tolerant (DST) maize to absorb moisture from subsoil zones is crucial to maize adaptation to water-stressed environments. The function of the mesocotyl in field emergence of seedlings is established in grasses. However, information is scarce on the extent of genetic variability for mesocotyl length (ML) in maize. Sixty-eight maize genotypes were studied using Completely Randomised Design in a laboratory experiment to investigate the extent of genetic variability for ML, and the relationship of seed biochemical components with ML. Ten seeds of each genotype were germinated for 10 days in the dark. Mesocotyl length was determined by placing cut mesocotyl against a flexible measuring tape. Biochemical contents of seeds were determined at a standard diagnostic laboratory. Analysis of variance revealed highly significant (p ≤ 0.01) genotype mean square, indicating sufficient variability for genetic improvement. Broad-sense heritability and genetic advance were high and implied that ML was heritable. Mean ML for genotypes ranged from 0.58 to 9.02 cm, thus, planned crosses can be made for ML improvement. A dendrogram from cluster analysis based on Ward’s minimum variance cluster analysis classified 65 of the genotypes into clusters I, II, and III with ML (mean ± standard deviation) of 0.49 ± 0.18, 4.25 ± 0.96, and 9.16 ± 0.93 cm, respectively. All the measured biochemical parameters, except selenium, showed significant (p ≤ 0.05/0.01) associations with ML. Crosses can be planned involving genotypes from clusters 1 and III, to exploit heterosis for ML in a hybrid program. The results obtained from this study provide a basis for the development of DST maize for drought-prone environments.