The soil-nitrogen condition, which differs greatly between paddy fields (mainly in the form of ammonium, NH₄⁺) and dry fields (mainly in the form of nitrate, NO₃^-), is a main environmental factor that drives the adaptive differentiation between upland and lowland rice ecotypes. However, the adaptive differentiation in terms of the nitrogen use efficiency (NUE) between upland and lowland rice has not been well addressed. In this study, we evaluated NUE-related traits among rice landraces as well as the genetic differentiation between NUE- associated genes and quantitative trait loci (QTLs). The japonica upland and lowland rice ecotypes showed large differences in their NUE-related traits such as the absorption ability for NH₄⁺ and NO₃^-. The indica upland and lowland rice exhibited similar performances when cultivated in solutions containing NH₄⁺ or NO₃^- or when planted in paddy or dry fields. However, the indica upland rice possessed a greater ability to absorb NO₃^-. We identified 76 QTLs for 25 measured traits using genome-wide association analysis. The highly differentiated NUE- associated genes or QTLs between ecotypes were rarely shared by japonica and indica subspecies, indicating an independent genetic basis for their soil-nitrogen adaptations. We suggested four genes in three QTLs as the candidates contributing to rice NUE during the ecotypic differentiation. In summary, the soil-nitrogen condition drives the adaptive differentiation of NUE between upland and lowland rice independently within the japonica and indica subspecies. These findings can strengthen our understanding of rice adaptation to divergent soil-nitrogen conditions and have implications for the improvement of NUE.