Carbon loss under high night-time temperature (HNT) leads to significant reduction in wheat yield. Growth chamber studies were carried out using six winter wheat genotypes, to unravel postheading HNT (23°C)-induced alterations in carbon balance, source-sink metabolic changes, yield, and yield-related traits compared with control (15°C) conditions. Four of the six tested genotypes recorded a significant increase in night respiration after 4 days of HNT exposure, with all the cultivars regulating carbon loss and demonstrating different degree of acclimation to extended HNT exposure. Metabolite profiling indicated carbohydrate metabolism in spikes and activation of the TriCarboxylic Acid (TCA) cycle in leaves as important pathways operating under HNT exposure. A significant increase in sugars, sugar-alcohols, and phosphate in spikes of the tolerant genotype (Tascosa) indicated osmolytes and membrane protective mechanisms acting against HNT damage. Enhanced night respiration under HNT resulted in higher accumulation of TCA cycle intermediates like isocitrate and fumarate in leaves of the susceptible genotype (TX86A5606). Lower grain number due to lesser productive spikes and reduced grain weight due to shorter grain-filling duration determined HNT-induced yield loss in winter wheat. Traits and mechanisms identified will help catalyze the development of physiological and metabolic markers for breeding HNT-tolerant wheat.