Improving the use of fertilizers is a major issue in sugarcane cropping system. Recent studies have highlighted the importance of considering the root component in assessing the crop nitrogen (N) requirements and the fertilizer-N use efficiency (NUE). A network of 11 trials was set up in Réunion to study the impact of fertilizer on root development in contrasting soil and climate conditions. Two fertilizer treatments (with optimal N fertilization (F) and without N fertiliser (NF) were investigated in each trial with at least three replicates. In each plot, root biomass and N concentration were assessed at harvest at three soil layers (0-10, 10-30 and 30-50 cm) and at three distances from the row. Nitrogen fertilization influenced the ratio of root to aboveground biomass significantly with an average increase in aboveground biomass of 17% and an average decrease in root biomass of 15% under fertilized conditions. N fertilization had no impact on the mass of N (Nmass) but increased the N concentration in the roots by 7%. The decrease in root biomass with N fertilization was mainly observed in the topsoil near the sugarcane row. The root biomass at harvest was strongly influenced by the soil type. With fertilization, the Nmass of the sugarcane roots represented 15 to 50% of the total Nmass accumulated in the entire sugarcane plant, depending on soil and climatic conditions. As a result, we suggest that this needs to be considered when determining the N requirements for sugarcane. The impact of soil and climate variability on root development and the influence of soil chemical characteristics on root development need further investigation.