Wheat is highly sensitive to heat stress, and even slight temperature fluctuations during critical growth stages can severely affect its growth, development, and yield. This sensitivity is further influenced by nitrogen availability and atmospheric CO₂ levels. The dynamic regulation of nitrate transporters in wheat under variable nitrogen, heat stress (HS), elevated CO₂ (eCO₂), and their combination plays a crucial role in modulating nitrate transport and plant adaptation, impacting crop productivity under changing environments. In this experiment, four Indian wheat cultivars (RAJ3765, PBW343, BT-Schomburgk, and Dharwad Dry) were subjected to heat stress (42 ± 2 °C), elevated CO₂ (650 ± 30 ppm), and their combination (HS + eCO₂) under three nitrogen regimes: nitrogen-deficient, nitrogen-optimum, and nitrogen-surplus. A putative NRT1 isoform (2476 bp) was cloned from the Dharwad Dry cultivar, sequenced, and submitted to GenBank (Accession no: OQ184870). In silico analysis revealed 12 transmembrane helices and a Major Facilitator Superfamily conserved domain, with an ORF of 1706 bp spanning from 338 to 2044 bp. Phosphorylation sites were enriched for threonine residues, suggesting a regulatory role in nitrate transport affinity. The cloned gene showed the highest homology to the NRT1/PTR FAMILY 6.4 gene of Aegilops tauschii subsp. strangulata. Maximum expression (3-fold upregulation) was observed in Dharwad Dry under elevated CO₂ at the pollination stage in root tissue under N-deficient conditions, while minimum expression was observed under heat stress in PBW343 under N-surplus conditions. Under heat stress conditions, RAJ3765 maintained better NRT1isofrom expression level compared to other cvs. These findings highlight the role of NRT1isoform in nitrogen use efficiency and stress adaptation, providing molecular targets for developing wheat cultivars better suited to future climate scenarios.