Due to the important role of cotton drought-tolerant varieties and the reported involvement in this trait of trehalose-6-phosphate-synthase, the respective gene (TPS) was isolated and characterized from cultivated cotton, Gossypium hirsutum (ZETA 2 cultivar), using a chromosome-walking technique. TPS has three exons comprising the coding region. Southern blot analysis indicated that the Gossypium genomes (A and D) contain a single copy of TPS per genome. In addition, the expression of this gene was studied in different plant tissues. Plants of the Australian cotton variety Siokra L23, known for its drought tolerance, were subjected to drought stress (using PEG 6,000 solution, for 4 h during the dark period of the day and for four consecutive days); leaves, stems and roots were collected after the end of the stress period. Total extracted RNA was examined for the presence of transcripts, in the above-mentioned tissues of stressed and well-watered plants, by reverse transcription-polymerase chain reaction (RT-PCR). The expression levels, determined semi-quantitatively, indicated that the gene was expressed in all plant tissues under both water availability conditions. However, increased expression levels of TPS were observed mainly in stressed leaves and roots compared to those of the well-watered control. This finding is in agreement with the fact that TPS participates in trehalose biosynthesis, known for its participation in stress signal transduction in higher plants.