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Citrullus colocynthis L. is one of the worldwide famous traditionally medicinal plants and widely applied in watermelon breeding for its multiple resistances. The complete nucleotide sequence of desert watermelon (Citrullus colocythis L.) chloroplast genome has been determined in this study. The genome was composed of 157,147 bp containing a pair of inverted repeats (IRs) of 26,149 bp, which was separated by a large single-copy region of 86,851 bp and a small single-copy region of 17,998 bp. A total of 123 genes were predicted including 86 protein-coding genes, eight rRNA genes and 29 tRNA genes. Phylogenetic analysis revealed that C. colocynthis were closely related to other two species in the genus Citrullus. The complete chloroplast genome of C. colocynthis would provide some significant information for Cucurbitaceae evolutionary and genomic studies.