Ammonium has been reported as a ‘preferred’ nitrogen (N) source for sugarcane (Saccharum spp.), which can improve N use efficiency (NUE) in this crop. We aimed to evaluate the preferential uptake of ammonium and nitrate in sugarcane genotypes contrasting with NUE under controlled conditions. Four sugarcane genotypes previously selected by another experiment (ER: efficient and responsive; ENR: efficient and nonresponsive; IR: inefficient and responsive; INR: inefficient and nonresponsive) were grown in a growth chamber and fertilized with two 15N-labeled forms [(NH4)2SO4 (15N-NH4+) or KNO3 (15N-NO3−)]; soil was used as substrate. Plants were evaluated at three time points: 0, 24, and 72 h after 15N-fertilization. For the efficient genotypes (ER and ENR), the soil NH4+ levels were about 20% lower than those found for the inefficient genotypes (IR and INR) indicating greater N extraction by the plant. Nitrogen derived from fertilizer (NDFF) and 15N recovery from fertilizer (15N RFF) in roots were influenced by the genotypes, in which responsive genotypes (ER and IR) presented a mean value 40% higher than the genotype INR, showing that greater absorption is more related to response than efficiency. Three days after N application, NDFF and 15N RFF from 15N-NH4+ were greater than 15N-NO3− in 40% and 65% for the roots and aerial part, respectively. The results of this study confirmed that sugarcane presents preferential uptake of NH4+ N form 3 days after fertilization. The use of nitrification inhibitors can be considered for providing a longer NH4+ residence time in the soil, also contributing to augmenting the NUE in sugarcane.