AbstractIn higher plants, Heat Shock Protein 20 (Hsp20) plays crucial roles in growth, development and responses to abiotic stresses. In this study, 94 GhHsp20 genes were identified in G. hirsutum, and these genes were phylogenetically clustered into 14 subfamilies. Out of these, 73 paralogous gene pairs remained in conserved positions on segmental duplicated blocks and only 14 genes clustered into seven tandem duplication event regions. Transcriptome analysis showed that 82 GhHsp20 genes were expressed in at least one tested tissues, indicating that the GhHsp20 genes were involved in physiological and developmental processes of cotton. Further, expression profiles under abiotic stress exhibited that two-thirds of the GhHsp20 genes were responsive to heat stress, while 15 genes were induced by multiple stresses. In addition, qRT-PCR confirmed that 16 GhHsp20 genes were hot-induced, and eight genes were up-regulated under multiple abiotic stresses and stress-related phytohormone treatments. Taken together, our results presented here would be helpful in laying the foundation for understanding the complex mechanisms of GhHsp20 mediated developmental processes and abiotic stress signaling transduction pathways in cotton.