Excessive use of nitrogen (N) in crops, such as potatoes, can lead to economic and environmental repercussions. We hypothesized that potato genotypes with resilient root systems and high genetic capabilities for nitrogen-use efficiency (NUE) could effectively mitigate these challenges. Consequently, we investigated intraspecific variations and characteristics within six distinct potato genotypes exhibiting diverse NUEs in response to varying nitrogen levels in an aeroponic system. The morpho-physiological and biochemical properties showed significant genotypic variations, especially related to the N-assimilating enzyme levels and root characteristics. Notably, the root systems of all genotypes demonstrated greater responsiveness to low nitrogen levels, with genotype C17 showcasing the most substantial root system irrespective of nitrogen concentration. Root morphological traits displayed robust positive correlations with NUtE, primarily influenced by genotype rather than nitrogen concentration. Conversely, nitrogen levels, displaying positive correlations with NUpE, influenced growth and activities of N-assimilating enzymes. Based on their distinct root systems, metabolic activities, and NUE profiles, genotypes C17 and C11 were determined to be N-efficient and N-inefficient, respectively. This study provides novel insights into the physiological and biochemical mechanisms underlying nitrogen use efficiency in potato genotypes under aeroponic conditions, offering potential targets for breeding programs, optimizing fertilizer management and cultivation strategies to improve crop performance under nitrogen-deficient conditions. Future investigations, employing multi-omics approaches, will elucidate key genes and pathways in nitrogen metabolism, potentially offering avenues to enhance root architecture and improve NUE.