Main conclusionThe chromosome-level genome assembly of Citrullus colocynthis reveals its genetic potential for enhancing drought tolerance, paving the way for innovative crop improvement strategies.This study presents the first comprehensive genome assembly and annotation of Citrullus colocynthis, a drought-tolerant wild close relative of cultivated watermelon, highlighting its potential for enhancing agricultural resilience to climate change. The study achieved a chromosome-level assembly using advanced sequencing technologies, including PacBio HiFi and Hi-C, revealing a genome size of approximately 366 Mb with low heterozygosity and substantial repetitive content. Our analysis identified 23,327 gene models, that could encode stress response mechanisms for species’ adaptation to arid environments. Comparative genomics with closely related species illuminated the evolutionary dynamics within the Cucurbitaceae family. In addition, resequencing of 27 accessions from the United Arab Emirates (UAE) identified genetic diversity, suggesting a foundation for future breeding programs. This genomic resource opens new avenues for the de novo domestication of C. colocynthis, offering a blueprint for developing crops with enhanced drought tolerance, disease resistance, and nutritional profiles, crucial for sustaining future food security in the face of escalating climate challenges.