<p>Potato Leafroll Virus (PLRV) is a devastating pathogen of potato that causes significant loss to crop production and productivity. This study highlighted the dynamics of morphological, physiological and biochemical responses in potato cultivars Kufri Karan and Kufri Pukhraj, which include its effect on tuber weight, plant height, carbohydrate metabolism, antioxidant defence systems, and enzymatic activities. Morphological observations were supplemented with reverse transcription polymerase chain reaction (RT-PCR), confirming the presence of PLRV in symptomatic plants. The number of tubers per plant and plant height were also significantly reduced, with the susceptible cultivar experiencing a greater decline. Carbohydrate analysis revealed reduced starch content, particularly in Kufri Pukhraj (45.1%), compared to a smaller reduction in Kufri Karan (21.8%). Both cultivars showed an increase in amylopectin, indicating a shift in carbohydrate metabolism due to stress. Enzyme activity related to carbohydrate breakdown, like α-amylase, β-amylase, and phosphorylase, increased to meet energy demands. In terms of antioxidant responses, carotenoid, phenol, and ascorbic acid levels increased under viral stress, particularly in Kufri Karan. The resistant cultivar exhibited enhanced enzymatic defences, with higher activities of catalase, peroxidase, and superoxide dismutase, contributing to its ability to mitigate oxidative stress caused by PLRV. The research provides valuable insights and mechanisms underlying PLRV with a specific focus on carbohydrate metabolism shifts, antioxidant defenses, and enzymatic activities, providing new insights into resistance mechanisms and stress adaptation in potatoes.</p> Graphical Abstract <p></p>