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Citrullus colocynthis (L.) Schrad, closely related to watermelon, is a member of the Cucurbitaceae family. This plant is a drought-tolerant species with a deep root system, widely distributed in the Sahara-Arabian deserts in Africa and the Mediterranean region. cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to study differential gene expression in roots of seedlings in response to a 20% polyethylene glycol-(PEG8000) induced drought stress treatment. Eighteen genes which show similarity to known function genes were confirmed by quantitative relative (RQ) real-time RT-PCR to be differentially regulated. These genes are involved in various abiotic and biotic stress and developmental responses. Dynamic changes with tissue-specific pattern were detected between 0 and 48 h of PEG treatment. In general, the highest induction levels in roots occurred earlier than in shoots, because the highest expression was detected in roots following 4 and 12 h, in shoots following 12 and 48 h of drought. These drought-responsive genes were also affected by the plant hormones abscisic acid (ABA), salicylic acid (SA), or jasmonic acid (JA), indicating an extensive cross-talk between drought and plant hormones. Collectively, these results will be useful to explore the functions of these multiple signal-inducible genes for unveiling the relationship and crosstalk between different signaling pathways.