Mesocotyl length (MES) is an important trait that affects the emergence of maize seedlings after deep-seeding and is closely associated with abiotic stress. The elucidation of constitutive-QTLs (cQTLs) and candidate genes for MES and tightly molecular markers are thus of great importance in marker-assisted selection (MAS) breeding. Therefore, the objective of this study was to perform detailed genetic analysis of maize MES across 346 F2:3 families, 30/30 extreme bulks of an F2 population, and two parents by conventional QTL analysis, bulked-segregation analysis (BSA), and RNA-sequencing when maize was sown at the depths of 3, 15, and 20 cm, respectively . QTL analysis identified four major QTLs in Bin 1.09, Bin 3.04, Bin 4.06–4.07, and Bin 6.01 under two or more environments, which explained 2.89–13.97% of the phenotypic variance within a single environment. BSA results revealed the presence of seven significantly linked SNP/InDel regions on chromosomes 1 and 4, and six SNP/InDel regions and the major QTL of qMES4-1 overlapped and formed a cQTL, cQMES4, within the 160.98–176.22 Mb region. In total, 18,001 differentially expressed genes (DEGs) were identified across two parents by RNA-sequencing, and 24 of these genes were conserved core DEGs. Finally , we validated 15 candidate genes in cQMES4 to involve in cell wall structure, lignin biosyntheis, phytohormones (auxin, abscisic acid, brassinosteroid) signal transduction, circadian clock, and plant organ formation and development. Our findings provide a basis for MAS breeding and enhance our understanding of the deep-seeding tolerance of maize.