The genetic basis of heterosis has not been resolved for approximately a century, although the role of loci with overdominant (ODO) effects has continued to be discussed by biologists. In the present investigation, a proposed model was studied in Gossypium hirsutum L. introgression lines (ILs) harbouring a segment of G. barbadense. These introgressions were confirmed by a single marker of G. barbadense. These ILs contained 396 quantitative trait loci (QTLs) for 11 yield and non-yield traits that were recorded in the field on homozygous and heterozygous plants for 5 years. After comparing the different types of QTLs between the yield group and the non-yield group, it was found that the yield group had significantly higher ODO QTL ratios. Moreover, 16 ODO QTLs identified for 5 yield-related traits were consistently detected during 5 cotton growing seasons (2010-2011 and 2013-2015): 6 of 7 for boll weight, 3 of 11 for seed-cotton yield per plant, 4 of 17 for boll number, 2 of 13 for lint yield per plant and 1 of 11 for lint percentage. Therefore, we propose that overdominance is the major genetic basis of lint yield heterosis in interspecific hybrids between G. barbadense and G. hirsutum. These findings have important implications in cotton breeding in that the boll weight can be improved by utilizing ODO QTLs via heterosis; thus, the stagnant yield barrier can be smashed to achieve sustainable increases in cotton production. Additionally, this concept can be translated to other field crops for improving their yield potential.