<p>Recent anthropogenic activities have spurred unparalleled abiotic environmental pressures, including drought, heat, cold, salinity, alkalinity, heavy metal stress. Among these stresses, salinity and alkalinity present substantial threats to agriculture, leading to poor crop yields, reduced food quality, economic losses, and challenges to food security and long-term agricultural sustainability. Elevated salt levels increase the production of reactive oxygen species (ROS) like superoxide anions (O₂^•−, hydrogen peroxide (H₂O₂), and hydroxyl radicals (^•OH), which can severely damage cell components like lipids, carbohydrates, proteins, and DNA. The study aimed to investigate the biochemical parameters of three red landraces of rice (<i>Oryza sativa</i> L.)‒ Karad, Jattoo, and Jhinjhan‒ to alkalinity-induced oxidative stress. Coloured crop landraces contain a plethora of antioxidants, have high nutritional value, and exhibit tolerance to a variety of stresses; hence, their utilization in agriculture can improve crop yields. The 4-day germinated rice seedlings were raised hydroponically and exposed to two different intensities of alkaline stress (25 and 50 mM NaHCO₃). After 4 days, roots and coleoptiles were evaluated for various parameters, including ROS (O₂^•−, H₂O₂), antioxidant assays (enzymatic and non-enzymatic antioxidants), and gene expression analysis to study the response to alkaline stress. Alkaline stress enhanced ROS production and lipid peroxidation, to a greater extent in the least alkaline-resistant landrace (Jhinjhan). Enzymatic antioxidants such as catalase, guaiacol peroxidase, ascorbate peroxidase, superoxide dismutase, monodehydroascorbate reductase, and dehydroascorbate reductase displayed higher activity in the stress-tolerant Karad landrace. The antioxidant enzyme activity increased with the highest stress dose, indicating that these enzymes play a crucial role in cellular defense against ROS and metabolites. In Karad, a significantly higher expression of anthocyanin biosynthetic pathway genes (chalcone synthase, phenylalanine ammonia-lyase, anthocyanidin synthase, and dihydroflavanol reductase) occurred, with over-representation of non-enzymatic antioxidants (proline, phenolics, ascorbate, non-protein thiols, and anthocyanins). Anthocyanin gene expression correlated well with the tissue anthocyanin concentration. Conversely, Jattoo and Jhinjhan showed less tolerance to alkaline stress in terms of biochemical parameters, together with the comparatively reduced expression of anthocyanin genes. Thus, the present study aims to offer valuable insights into the alkaline stress tolerance of red rice landraces, which could be related to the augmented responsiveness of antioxidant machinery and constitutive/induced anthocyanin genes, resulting in more efficient anthocyanin accumulation. As research advances, understanding the full potential of modern techniques, such as developing gene-specific molecular markers from coloured rice, genetic engineering, and genomic approaches, is crucial to increasing the frequency of desired traits in breeding populations, especially those designed for deployment in target environments.</p>