Abstract: Whole genome duplication, a prevalent force of evolution in plants, results in massive genome restructuring in different organisms. Roles of the resultant duplicated genes are poorly understood, both functionally and evolutionarily. In the present study, differentially expressed ethylene responsive factors (GhERF1s), anchored on Chr-A07 and Chr-D07, were isolated from a high-yielding cotton hybrid (XZM2) and its parents. The GhERF1 was located in the B3 subgroup of the ethylene responsive factors subfamily involved in conferring tolerance to abiotic stress. Nucleotide sequence analysis of 524 diverse accessions, together with quantitative real-time polymerase chain reaction analysis, elucidated that de-functionalization of GhERF1-7A occurred due to one base insertion following formation of the allotetraploid cotton. Our quantitative trait loci and association mapping analyses highlighted a role for GhERF1-7A in conferring high boll number per plant in modern cotton cultivars. Overexpression of GhERF1-7A in transgenic Arabidopsis resulted in a substantial increase in the number of siliques and total seed yield. Neo-functionalization of GhERF1-7A was also observed in modern cultivars rather than in races and/or landraces, further supporting its role in the development of high-yielding cotton cultivars. Both de- and neo-functionalization occurred in one of the duplicate genes, thus providing new genomic insight into the evolution of allotetraploid cotton species.