BACKGROUND: In plants, FK506-binding proteins (FKBPs) have been shown to participate in various biological processes such as photosynthetic system reaction, stress response, and growth and development. However, the roles of FKBPs in cotton are less well known. RESULTS: In this study, we investigated FKBP family genes on a genome-wide scale in four Gossypium species. A total of 147 FKBP genes were identified from these four Gossypium species and placed into three classes based on phylogenetic analysis. Collinearity analysis indicated that whole-genome duplication events and segmental duplication events were the main sources of gene amplification during the evolution of FKBP genes. Conserved motif, expression profiles and cis-acting elements prediction of the GhFKBPs analysis revealed that GhFKBPs were differentially expressed in different tissues and under abiotic stress. qRT-PCR analysis showed that some GhFKBPs were predominantly expressed in leaves. The analysis of cis-acting elements prediction revealed that MYB, MYC and ERE related binding sites in the promoters of GhFKBP genes were the most abundant. Furthermore, the composition and distribution of these cis-acting elements exhibited differences between homologous GhFKBP gene pairs. Silencing of GhFKBP13 in cotton resulted in disruption of chloroplast structure and starch metabolism disorders. CONCLUSIONS: Taken together, 147 FKBP family genes in four Gossypium species are comprehensively characterized, and GhFKBP13 play a critical role in chloroplast biogenesis in upland cotton.