Comparative transcriptome analysis of fiber tissues betweenGossypium barbadenseandGossypium hirsutumcould reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage inG. barbadense, which showed four distinct expression patterns (I–IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis betweenG. barbadenseandG. hirsutum. Subsequently, we overexpressedGbAAR3andGbTWS1, two of the 42 candidate genes, inArabidopsisplants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I–V) inG. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from theirG. hirsutumorthologs, and the genotyping and phenotyping of a BC3F5population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences betweenG. barbadenseandG. hirsutumand provide novel alternative genes for fiber quality improvement.