The nutritional value of cucumber (Cucumis sativus L.) can be improved by the introgression of β-carotene (i.e., provitamin A and/or orange flesh) genes from “Xishuangbanna gourd” (XIS; Cucumis sativus var. xishuangbannanesis Qi et Yuan) into US pickling cucumber. However, the genetics of β-carotene content has not been clearly defined in this US market type. Thus, three previous populations derived from a US pickling cucumber (‘Addis’) × XIS mating were evaluated for β-carotene content, from which the high β-carotene inbred line (S₄), ‘EOM 402-10’, was developed. A cross was then made between the US pickling cucumber inbred line ‘Gy7’ [gynoecious, no β-carotene, white flesh; P₁] and ‘EOM 402-10’ [monoecious, possessing β-carotene, orange flesh; P₂] to determine the inheritance of β-carotene in fruit mesocarp and endocarp tissue. Parents and derived cross-progenies (F₁, F₂, BC₁P₁, and BC₁P₂) were evaluated for β-carotene content in a greenhouse in Madison, Wisconsin. While F₁ and BC₁P₁ progeny produced mature fruits possessing white, light-green, and green (0.01–0.02 μg g⁻¹ β-carotene) mesocarp, the F₂ and BC₁P₂ progeny mesocarp segregated in various hues of white, green, yellow (0.01–0.34 μg g⁻¹ β-carotene), and orange (1.90–2.72 μg g⁻¹ β-carotene). Mesocarp and endocarp F₂ segregation adequately fit a 15:1 [low-β-carotene (0.01–0.34 μg g⁻¹): high-β-carotene (1.90–2.72 μg g⁻¹)] and 3:1 (low-β-carotene: high-β-carotene) ratio, respectively. Likewise, segregation of carotene concentration in mesocarp and endocarp tissues in BC₁P₂ progeny adequately fit a 3:1 (low-β-carotene: high-β-carotene) and 1:1 (low-β-carotene: high-β-carotene) ratio, respectively. Progeny segregations indicate that two recessive genes control the β-carotene content in the mesocarp, while one recessive gene controls β-carotene content in the endocarp. Single marker analysis of F₂ progeny using the carotenoid biosynthesis gene Phytoene synthase determined that there was no association between this gene and the observed β-carotene variation in either fruit mesocarp or endocarp.