The single-celled cotton (Gossypium hirsutum) fiber provides an excellent model to investigate how human selection affects phenotypic evolution. To gain insight into the evolutionary genomics of cotton domestication, we conducted comparative transcriptome profiling of developing cotton fibers using RNA-Seq. Analysis of single-celled fiber transcriptomes from four wild and five domesticated accessions from two developmental time points revealed that at least one-third and likely onehalf of the genes in the genome are expressed at any one stage during cotton fiber development. Among these, ,5,000 genes are differentially expressed during primary and secondary cell wall synthesis between wild and domesticated cottons, with a biased distribution among chromosomes. Transcriptome data implicate a number of biological processes affected by human selection, and suggest that the domestication process has prolonged the duration of fiber elongation in modern cultivated forms. Functional analysis suggested that wild cottons allocate greater resources to stress response pathways, while domestication led to reprogrammed resource allocation toward increased fiber growth, possibly through modulating stress-response networks. This first global transcriptomic analysis using multiple accessions of wild and domesticated cottons is an important step toward a more comprehensive systems perspective on cotton fiber evolution. The understanding that human selection over the past 5,000+ years has dramatically re-wired the cotton fiber transcriptome sets the stage for a deeper understanding of the genetic architecture underlying cotton fiber synthesis and phenotypic evolution.