Cotton productivity is affected by water deficit, and little is known about the molecular basis of drought tolerance in cotton. In this study, microarray analysis was conducted to identify drought-responsive genes in the third topmost leaves of the field-grown drought-tolerant cotton (Gossypium hirsutum L.) cultivar Acala 1517-99 under drought stress conditions. Water stress was imposed by withholding irrigation for 9 days in the early squaring stage, which resulted in 10-15 % reduction in plant growth compared to the well-watered plants. A total of 110 drought-responsive genes (0.5 % of the total genes) were identified, 79 % (88 genes) of which were drought-repressed and 21 % (22 genes) were drought-induced. The drought-induced genes were grouped into six functional categories including stress-related (ten genes, nine of which encode heat shock proteins), metabolism (three genes) and one gene each for transcription factor, proline biosynthesis and cellular transport. The drought-repressed genes were classified into 14 functional categories, comprising metabolism (20 genes), cellular transport (12 genes), stress-related (12 genes), regulation of gene expression (nine genes), transcription factor (four genes), signal transduction (seven genes) and two genes each for biosynthesis of secondary compounds, cell wall, fatty acids/lipids and chlorophyll, and protein degradation. Most of the genes have been reported in other plants as drought-tolerant/responsive. The responsiveness of 19 selected drought-responsive genes was validated by quantitative RT-PCR. Furthermore, primers were developed and assayed for all the drought-responsive genes to develop single-strand conformation polymorphic markers, many of which were found to be correlated with drought tolerance. This report represents the first study on integration of a transcriptome analysis to develop molecular markers that are associated with drought tolerance in cotton.