Cotton breeders face many problems, such as negative association between lint yield and fiber strength, in their efforts to improve fiber quality through conventional breeding methods. The primary goal of this project was to identify and characterize genes at the molecular level related to cotton fiber development. Messenger RNAs were isolated from fibers at 20 day-post-anthesis (dpa) of a high yielding cotton accession TM-1 (Gossypium hirsutum L.) and of a high fiber quality cotton accession 3-79 (G. barbadense L). A cDNA library was constructed from mRNA obtained from cotton fiber (20 dpa) of 3-79 and screened sequentially with cDNA probes from TM-1 and 3-79. Thirteen cDNAs were identified. Five of the thirteen cDNAs (GbFb1, GbFb2, GbFb3, GbMAPK and GbLTP) were highly expressed in the 20-dpa fiber tissue of 3-79 cotton. Sequence analysis indicated a cotton lipid transfer protein (GbLTP), a mitogen-activated protein kinase (GbMAPK), and a novel gene with no homologous sequence (GbFb1) in the GenBank database. The remaining two cDNAs with unknown functions, GbFb2 and GbFb3, showed high identity to fiber ESTs from G. hirsutum at 6 d and G. arboreum L. at 7 to 10 dpa, respectively. These five genes are developmentally regulated, and they showed much higher expression levels in the 20-dpa fiber than in the other tissues tested, except the gene for GbFb1, which was abundantly expressed in the flower. These results are among the few reports of putative gene expression associated with fiber in G. barbadense.