With increasing population, degrading soil health, limited arable land area, and high cost of nitrogen (N) fertilizers, improving nitrogen use efficiency (NUE) of potato is an inevitable approach to save the environment and achieve sufficient tuber yields with less N fertilizer supply. Recently, we have developed an aeroponics system to study NUE in potato using genomics, physiology, and breeding approaches. This study aims on precision phenotyping of plants of two distinct potato varieties (Kufri Gaurav, N efficient; Kufri Jyoti, N inefficient) in the novel aeroponics system. Plants were grown in aeroponics under controlled conditions with low N (0.75 mmol L–1 NO3 –) and high N (7.5 mmol L–1 NO3 –) levels. Plant biomass, root traits, total chlorophyll content, and plant N were increased with increasing N supply, whereas higher NUE parameters namely NUE, agronomic NUE (AgNUE), N uptake efficiency (NUpE), harvest index (HI), and N harvest index (NHI) were observed at low N. An NUE efficient cv. Kufri Gaurav showed higher tuber dry weight, fresh tuber yield, tuber number per plant, early start of tuber harvesting, root traits, stolon traits, NUE parameters, and higher amino acid (aspartic acid and asparagine) content at low N supply. Higher expression of nitrate reductase (NR), nitrite reductase (NIR), and asparagine synthetase (AS) genes was observed in the leaf tissues of Kufri Gaurav at high N. Thus, aeroponics-based precision phenotyping enables identification of NUE efficient genotypes based on key traits and genes involved in improving NUE in potato. Further, this study suggests that the potential of aeroponics can be utilized to investigate N biology in potato under different N regimes.