Fasciclin-like arabinogalactan (FLA) protein is a cell-wall-associated protein playing crucial roles in regulating plant growth and development, and it was characterized in different plants including Upland cotton (Gossypium hirsutum L.). In cDNA-AFLP analysis of 25 DPA (days post anthesis) fiber mRNA, two FLA gene-related transcripts exhibit differential expression between Sea Island cotton (G. barbadense L.) and Upland cotton. Based on the transcript-derived fragment, RACE-PCR and realtime PCR technique, GbFLA5 full-length cDNA was isolated and its expression profiles were characterized in both cotton plant tissues and secondary cell wall (SCW) fibers in this study. The 1154 bp GbFLA5 cDNA contains an ORF of 720 bp, encoding GbFLA5 protein of 239 amino acids residues in length with an estimated molecular mass of 25.41 kDa and isoelectric point of 8.63. The deduced GbFLA5 protein contains an N-terminal signal sequence, two AGP-like domains, a single fasciclin-like domain, and a GPI anchor signal sequence. Phylogenetic analysis shows that GbFLA5 protein is homologous to some known SCW-specific expressed FLAs of plant developing xylem, tension wood and cotton fibers. In the SCW deposition stage from 15 to 45 DPA detected, FLA5 maintains a significantly higher expression level in Sea Island cotton fibers than in Upland cotton fibers. The increasing FLA5 transcript abundance coincided with the SCW deposition process and the expression intensity differences coincided with their fiber strength differences between Sea Island cotton and Upland cotton. These expression profile features of GbFLA5 in cotton fibers revealed its tissue-specific and SCW developmental stage-specific expression characters. Further analysis suggested that GbFLA5 is a crucial SCW-specific protein which may contribute to fiber strength by affecting cellulose synthesis and microfibril deposition orientation.