Class III homeodomain-leucine zipper (HD-Zip III) genes are important plant-specific transcription factors which have key roles in different stages of vascular and interfascicular fiber differentiation. A novel HD-Zip III gene, designated GbHB1, was isolated by suppression subtraction hybridization and RACE (rapid amplification of cDNA ends) from Gossypium barbadense (sea-island cotton). The GbHB1 cDNA has a total length of 3061 bp with an open reading frame of 2508 bp, encoding a predicated polypeptide of 836 amino acids with a molecular weight of 91.6 kDa and a calculated pI of 5.93. The putative polypeptide of GbHB1 is structurally characterized by a homeodomain positioned adjacent to a leucine zipper domain, which shares high identity with other reported HD-Zip III domains. DNA gel blotting analysis shows that GbHB1 is a low-copy gene. Organ expression pattern analysis reveals that GbHB1 expressed highly in ovule and stem, followed by in root, and low in leaf and cotyledon. The result suggests that GbHB1 may play a regulatory role in cotton interfascicular fiber development.