Cassava (Manihot esculenta) is a key food security crop, providing a fundamental carbohydrate source for hundreds of millions of people globally. Recent initiatives aim to increase the provitamin A (PVA) carotenoid content of cassava roots to combat the high rates of vitamin A deficiency in sub-Saharan Africa, where cassava is a staple food. However, these breeding efforts have been hindered by a negative genetic correlation between total carotenoid (TC) and dry matter (DM) contents. We investigated the genetic and physiological mechanisms underlying this trade-off, providing insights to guide breeding strategies that prioritize both nutritional and agronomic traits. In the first chapter, four potential mechanisms are discussed: competition for precursors, plastid interactions, signaling influences, and statistical correlations. The second chapter investigates a 10 Mbp introgression from the wild relative Manihot glaziovii on cassava chromosome 1, which has been thought to contribute to maintaining a hypothesized repulsion linkage driving the trade-off between TC and DM. We developed a panel of 10 KASP markers to tag the glaziovii haplotype and select recombinants to comprise a mapping population of 500 lines. Quantitative trait loci (QTL) for aboveground vigor were mapped with the aim to identify deleterious effects of the introgression. The third chapter is a bivariate genome-wide association study (GWAS) to examine the genetic architecture of the TC-DM relationship. Using a population of 372 yellow cassava accessions, this study identified 17 loci jointly associated with TC and DM and found significant residual polygenic covariance, supporting the hypothesis of genome-wide mediated pleiotropy. The fourth chapter integrates transcriptomic and metabolomic data to analyze interactions between carotenoids and carbohydrate metabolism across genetic, spatial, and developmental variation. Carotenoid composition analysis suggested that cis-carotene abundance was more important than TC in driving the negative correlation with DM. Gene-metabolite networks across both genetic and non-genetic variation were constructed, revealing that carotenoid-related and starch-related genes are not globally co-regulated, though individual associations between starch-related genes and carotenoid metabolites were characterized. Together, this research advances fundamental understanding of the genetic and physiological interactions between carotenoid and dry matter contents to help guide breeding strategies for cassava biofortification.