A conventional breeding program was established to transfer the bacterial phytoene synthase transgene-crtB-from a transgenic, white-rooted cassava to yellow-rooted cassava plants carrying the endogenous phytoene synthase alleles named psy2-y and/or psy2-y. Combining endogenous phytoene synthase enzymes (PSYs) with CRTB in a single cassava plant would allow the molecular dissection of individual allele contributions to carotenoid synthesis and/or accumulation in cassava roots. The simultaneous expression of the crtB transgene and psy2-y in individuals planted in the field coincided with higher total, HPLC-quantified carotenoid content in roots, although the variability among replications (plants) precluded the detection of statistically significant differences. Nevertheless, the highest total carotenoid content in roots within a family coincided with one individual of the F1 progeny carrying both psy2-y and crtB genes. The results also indicated the presence of at least one more key gene-different from psy or crtB-which too is necessary for the synthesis and/or accumulation of Pro-Vitamin A carotenoids in cassava roots.