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Background Improving the yield and fiber quality of upland cotton is a goal of plant breeders. However, increasing the yield and quality of cotton fibers is becoming more urgent. While the growing human population needs more cotton fiber, climate change is reducing the amount of land on which cotton can be planted, or making it difficult to ensure that water and other resources will be available in optimal quantities. The most logical means of improving yield and quality is understanding and manipulating the genes involved. Here, we used comparative transcriptomics to explore differences in gene expression between long- and short-fiber cotton lines to identify candidate genes useful for cotton improvement. Results Light and electron microscopy revealed that the initial fiber density was significantly greater in our short-fiber group (SFG) than in our long-fiber group (LFG). Compared with the SFG fibers, the LFG fibers were longer at all developmental stages. Comparison of the LFG and SFG transcriptomes revealed a total of 3538 differentially expressed genes (DEGs). Notably, at all three developmental stages examined, two expression patterns, consistently downregulated (profile 0) and consistently upregulated (profile 7), were identified, and both were significantly enriched in the SFG and LFG. Twenty-two DEGs known to be involved in fiber initiation were detected in profile 0, while 31 DEGs involved in fiber elongation were detected in profile 7. Functional annotation suggested that these DEGs, which included ERF1, TUA2, TUB1, and PER64, affect fiber elongation by participating in the ethylene response, microtubule synthesis, and/or the peroxidase (POD) catalytic pathway. qRT-PCR was used to confirm the RNA sequencing results for select genes. Conclusions A comparison of SFG and LFG transcription profiles revealed modest but important differences in gene expression between the groups. Notably, our results confirm those of previous studies suggesting that genes involved in ethylene, tubulin, and POD pathways play important roles in fiber development. The 22 consistently downregulated DEGs involved in fiber initiation and the 31 consistently upregulated genes involved in fiber elongation are seemingly good candidate genes for improving fiber initiation and elongation in cotton.