UDP-xylose (UDP-Xyl) is a nucleotide sugar used as substrate for producing non-cellulose materials. e. g., hemicellulose and pectic polysaccharide, in the fibers of cotton (Gossypium). Its biosynthesis is catalyzed from UDP-glucuronic acid (UDP-GlcA) by UDP-glucuronate decarboxylase (UXS). Here, we first cloned GhUXS1 in Gossypium hirsutum and Gossypium barbadense based on a transcript-derived fragment that originated from our cDNA-AFLP transcriptome profiling in cotton. GhUXS2 and GhUXS3 were also isolated via homology-based cloning. GhUXS nucleotide sequences were identical between the two species, and the deduced amino acid residues had the conserved motif of the UXS family, i.e., GxxGxxG, Ser residues, and YxxxK. GhUXS1 was expressed in Escherichia coli and generated UXS activity that converted UDP-GlcA to UDP-Xyl. Semiquantitative RT-PCR results showed that GhUXSs transcripts were preferentially expressed during fiber development, from elongation through the stage of secondary cell wall synthesis. Although at the same number of days post-anthesis they were more abundant in G. hirsutum, expression was sustained for a longer period in G. barbadense. These different patterns of expression may affect quality and partially explain why the latter species has better fiber strength.