Key message: β-Carotene content in sweetpotato is associated with the Orange and phytoene synthase genes; due to physical linkage of phytoene synthase with sucrose synthase, β-carotene and starch content are negatively correlated. Abstract: In populations depending on sweetpotato for food security, starch is an important source of calories, while β-carotene is an important source of provitamin A. The negative association between the two traits contributes to the low nutritional quality of sweetpotato consumed, especially in sub-Saharan Africa. Using a biparental mapping population of 315 F1 progeny generated from a cross between an orange-fleshed and a non-orange-fleshed sweetpotato variety, we identified two major quantitative trait loci (QTL) on linkage group (LG) three (LG3) and twelve (LG12) affecting starch, β-carotene, and their correlated traits, dry matter and flesh color. Analysis of parental haplotypes indicated that these two regions acted pleiotropically to reduce starch content and increase β-carotene in genotypes carrying the orange-fleshed parental haplotype at the LG3 locus. Phytoene synthase and sucrose synthase, the rate-limiting and linked genes located within the QTL on LG3 involved in the carotenoid and starch biosynthesis, respectively, were differentially expressed in Beauregard versus Tanzania storage roots. The Orange gene, the molecular switch for chromoplast biogenesis, located within the QTL on LG12 while not differentially expressed was expressed in developing roots of the parental genotypes. We conclude that these two QTL regions act together in a cis and trans manner to inhibit starch biosynthesis in amyloplasts and enhance chromoplast biogenesis, carotenoid biosynthesis, and accumulation in orange-fleshed sweetpotato. Understanding the genetic basis of this negative association between starch and β-carotene will inform future sweetpotato breeding strategies targeting sweetpotato for food and nutritional security.