Maize (Zea mays L) is the most important food grain in sub-Saharan Africa and is mostly grown by small-scale farmers under rainfed conditions. Aluminum toxicity caused by low pH is one of the abiotic factors limiting maize production among smallholder farmers. Therefore, breeding maize hybrids that are tolerant to aluminum toxicity will sustain and increase maize production in these areas. Hence this study was undertaken to assess the genotypic variation for aluminum toxicity in maize inbred lines. Fourteen maize inbred lines of historical importance that are used in maize hybrid breeding in Zambia were studied for seedling root variation under different aluminum concentrations using hydroponic conditions. The aluminum tolerance membership index based on three traits (actual root length, relative root length and root length response) classified genotypes L3233 and L1214 as highly tolerant, L5527 and ZM421 as tolerant, and L12, L3234, and ZM521 as intermediate. The high PCV, GCV, and heritability observed for the root traits indicate that opportunities for selection and breeding for aluminum tolerance among Zambian inbred lines exist. Furthermore, the study indicated that a higher genetic gain would be expected from net root growth followed by shoot length response as selection traits, thus supporting the use of root traits for aluminum tolerance screening. OPEN ACCESSAgronomy 2015, 5 201