A better knowledge of genetic variability of traits related to nitrogen use efficiency (NUE) is a potential strategy to optimize N fertilization and to reduce environmental pollution without decreasing marketable yield and quality. To this aim, in this study, 13 cultivars of witloof chicory were compared with three reference cultivars known for their adaptation to low, intermediate and high N availability in the field during the vegetative phase of development. Pertinent criteria used for this study were determined by a thorough comparison of nitrogen reserve accumulation in tuberized roots during vegetative development and mobilization during the forcing process in the three reference cultivars. Cluster analysis allowed us to sort the cultivars into four main groups we named G1, G2, G3 and G4. Cultivars of group G4, better adapted to soils with high nitrogen contents (N-demanding cultivars), showed higher total N, nitrate, total amino acids (AA), glutamine contents and lower total N and AA mobilization for chicon growth than did cultivars of group G1, adapted to soils with low nitrogen content (N-sensitive cultivars). An intermediate behavior was exhibited by cultivars of groups G2 and G3, characterized as N tolerant. It is proposed that either chicory growers or breeders may take advantage of the genetic variability revealed in the present study to gain flexibility in choosing the right cultivar for the type of soil available (N-rich soil vs N-poor soil) or to adapt the level of N fertilization to the type of cultivar (N-demanding vs N-sensitive) in order to target the highest NUE for the best chicon yield and trade quality.