MYB family transcription factors are implicated in multiple developmental processes. Herein, a new full-length cDNA encoding a SANT/MYB transcription factor (designated as GbRL2) was cloned and characterized from cotton (Gossypium barbadense L.) for the first time. The full-length cDNA of GbRL2 was 573 bp with a 240 bp open reading frame (ORF) encoding a deduced protein of 80 amino acid polypeptide with a calculated molecular mass of 8.96 kDa and an isoelectric point of 8.96. Sequence alignment revealed that GbRL2 had high homology with other single SANT/MYB domain containing genes, including the RADIALIS genes in Antirrhinum majus and Bournea leiophylla. Semi-quantitative reverse transcript polymerase chain reaction (RT-PCR) revealed that at seedling stage, GbRL2 was strongly expressed in leaves but merely in stems. In opening flowers, the expression of GbRL2 was moderate in the petals but could not be detected in stamens. In ovules, the expression of GbRL2 could not be detected at -3 days post-anthesis (DPA) but increased during early elongation stage (0 DPA, +3 DPA, +5 DPA and +8 DPA). The transcripts of GbRL2 could also be detected at +8 DPA elongating fibers. We also examined the expression of RL2 gene in Gossypium hirstum cultivar Xu-142 and its fuzzless-lintless-seed mutant fl plants. The GhRL2 gene was ectopically expressed at -3 DPA in the fl mutant while the expression of GhRL2 in WT could not be detected. The expression of GhRL2 decreased early (+5 DPA) while that of WT was still strong. Our results suggest that GbRL2 may participate in development of various organs and may be a target for genetic improvement of cotton fiber.