The tuberous root crop, yacon, is native to the Andean region and contains high amountsof fructooligosaccharides (FOS) with up to 70% of dry matter. Due to FOS, consumption of tubersmay have health-promoting effects. However, regarding the overall cultivation system, norecommendations exist for farmers on nitrogen fertilization and nitrogen (N) uptake of yacon.Therefore, three different N fertilization levels (0, 40, and 80 kg N ha−1) and two genotypes (brownshelled(BG) and red-shelled (RG)) were examined in a two-year field experiment regarding theirtuber yield, sugar composition, and nitrogen uptake. Tuber yields increased with increasingfertilization level and were highest for B80 and R80 (50 and 67 t FM ha−1), while significantdifferences between the genotypes existed. Sugar and the amount of FOS slightly decreased withincreasing N fertilization level, and ranged between 36% and 73% and 30% and 58% of dry matter,respectively. An overall decreasing amount of FOS led to a slight increase in the amount of FOSwith a higher degree of polymerization. Regarding the N-use efficiency of tubers and the total plant,an N fertilization level of 40 kg N ha−1 seems to favor tuber yield.