Cassava is an important crop for millions of people, particularly those living in marginal agriculture land in tropical and subtropical regions of the world. Cassava is an excellent source of calories, but enhancement of the nutritional quality, including carotenoids, minerals and proteins is highly desirable. For efficient improvement of carotenoids content in segregating cassava populations, proper sampling procedures are needed. For this study, roots from cloned plants of genotype CM 2772-3 were harvested and analyzed. Samples along the longitudinal (proximal, central and distal section) and across the transversal (periphery, mid-parenchyma and core) axes of the roots were analyzed. Plant-to-plant variation was only significant for dry matter content. Carotenoids and dry matter content were quantified individually in 243 root samples. Carotenoids content (fresh weight basis) was higher in the proximal sections and gradually lower in the central and distal portions. An opposite trend was observed when carotenoids were quantified on dry weight basis. Carotenoids concentrations were higher in the core and lower towards the periphery of the root (both fresh and dry weight basis). A proper sampling methodology to overcome differences according to the results of this study is proposed.