Cucumis hystrix Chakr. (2n = 2x = 24) is a wild species that can hybridize with cultivated cucumber (C. sativus L., 2n = 2x = 14), a globally important vegetable crop. However, cucumber breeding is hindered by its narrow genetic base. Therefore, introgression from C. hystrix has been anticipated to bring a breakthrough in cucumber improvement. Here, we report the chromosome-scale assembly of C. hystrix genome (289 Mb). Scaffold N50 reached 14.1 Mb. Over 90% of the sequences were anchored onto 12 chromosomes. A total of 23,864 genes were annotated using a hybrid method. Further, we conducted a comprehensive comparative genomic analysis of cucumber, C. hystrix, and melon (C. melo L., 2n = 2x = 24). Whole-genome comparisons revealed that C. hystrix is phylogenetically closer to cucumber than to melon, providing a molecular basis for the success of its hybridization with cucumber. Moreover, expanded gene families of C. hystrix were significantly enriched in "defense response," and C. hystrix harbored 104 nucleotide-binding site-encoding disease resistance gene analogs. Furthermore, 121 genes were positively selected, and 12 (9.9%) of these were involved in responses to biotic stimuli, which might explain the high disease resistance of C. hystrix. The alignment of whole C. hystrix genome with cucumber genome and self-alignment revealed 45,417 chromosome-specific sequences evenly distributed on C. hystrix chromosomes. Finally, we developed four cucumber-C. hystrix alien addition lines and identified the exact introgressed chromosome using molecular and cytological methods. The assembled C. hystrix genome can serve as a valuable resource for studies on Cucumis evolution and interspecific introgression breeding of cucumber.