Most cucumber varieties have fruits with white flesh, which is devoid of ß-carotene and has a low concentration of total carotenoids. Carotenoids are important nutrients for humans and animals. Thus, developing cucumber varieties with orange flesh could provide a new nutritionally enhanced food source. Some cucumbers with yellow and orange flesh have been described, but there are others that have not been studied. Here, we used three cucumber PI lines, reported to produce colored fruits, from the USDA National Plant Germplasm System to generate three F2 populations. Fruits from the F2 populations with colored flesh (green, yellow, or orange) were pooled, and an equal number of fruits with white flesh were pooled. RNA was isolated from the pools and used for RNA sequencing to determine gene expression differences and to identify SNPs in each pool. The orange color of the cucumber fruits was confirmed to be due to ß-carotene. There were no clear expression patterns for genes of the carotenoid biosynthesis pathway that would suggest that their expression controlled the coloration of fruits. Mutations in carotenoid biosynthesis genes also did not explain the variation. However, we detected a SNP in the homolog of the Or gene that is responsible for ß-carotene accumulation in orange cauliflower. This genetic basis is different from that of previously studied orange cucumbers, but our results suggest that Or is not the only factor. These results provide the basis for future studies for breeding orange cucumbers for commercial or home garden production.