Technical Abstract: Potato (Solanum tuberosum L.) tubers contain a wide range of carotenoid content. To decipher the key factors controlling carotenoid levels in tubers, four potato lines (Atlantic, Désirée, 91E22, and POR03) were examined by a combination of biochemical, molecular, and genomics approaches. These lines contained incremental levels of carotenoids, which were found to be associated with enhanced capacity of carotenoid biosynthesis as evident from norflurazon treatment. Microarray analysis of high and low carotenoid lines (POR03 vs. Atlantic) revealed 381 genes that showed significantly differential expression. The carotenoid metabolic pathway genes Beta-carotene hydroxylase 2 (BCH2) and Beta-carotene hydroxylase 1 (BCH1), along with zeaxanthin epoxidase (ZEP), and carotenoid cleavage dioxygenase 1A (CCD1A) were among the most highly differentially expressed genes. The transcript levels of BCH2 and BCH1 were lowest in Atlantic and highest in POR03, whereas those of ZEP and CCD1A were high in low carotenoid lines and low in high carotenoid lines. The high expression of BCH2 in POR03 line was associated with enhanced response to sugars. Our results indicate that high levels of carotenoid accumulation in potato tubers were due to an increased metabolic flux into the carotenoid biosynthetic pathway, as well as the differential expression of carotenoid metabolic genes.