Worldwide, the nitrogen use efficiency (NUE) for crop plants is of great concern. The burgeoning world population needs crop genotypes that respond to higher nitrogen and show a direct relationship to yield with use of nitrogen inputs, i.e. high nitrogen-responsive genotypes. However, for fulfilling the high global demand of organic produce, it requires the low nitrogen responsive genotypes with greater NUE and grain yields. The lack of knowledge about precise regulatory mechanisms to explain NUE in crop plants hampers the goal of agricultural productivity. Understanding the molecular basis of NUE will enable to provide handle for crop improvement through biotechnological means. With the advent of modern genomics and proteomics approaches such as subtractive hybridization, differential display, and microarray techniques are revolutionizing to identify the candidate genes which play a pivotal role in the regulation of NUE. Beside it, quantitative real-time polymerase chain reaction technology is also being used to establish marker-trait association for NUE. The identification of potential candidate genes/proteins in the regulation of NUE will serve as biomarker(s) for screening genotypes for their nitrogen responsiveness for optimization of nitrogen input in agriculture. This paper describes the molecular basis of NUE with respect to nitrogen metabolism and its intimate relationship with carbon metabolism, use of molecular-physiological-genetics approaches for understanding the role of various genes/proteins, and their validation to use as biomarker(s) for determining genotypic potential for NUE. Since NUE in plants is a complex trait which not only involves the primary process of nitrogen uptake and assimilatory pathways but also a series of events, including metabolite partitioning, secondary remobilization, C-N interactions, as well as molecular signalling pathways and regulatory control outside the metabolic cascades. Therefore, identification of novel nitrogen responsive genes and their cis- and trans-acting gene elements is essential. Thus, fishing out a single gene, biomarker or a master regulator controlling complex trait of NUE could serve as an appropriate strategy for nitrogen management in agriculture.