Heat stress is a prime constraint hampering the attainment of cotton yield potential in Pakistan, while selection of genotypes for high temperature environment using morphological markers often leads to discrepancies. An experiment was conducted in order to characterize thermo-sensitivity of varying cotton genotypes using biochemical markers and to determine the biochemical attributes modulated regulations in biomass accumulation of heat-stressed cotton. The experiment was replicated thrice and laid out in completely randomized design (CRD) with split arrangement during 2016 at the University of Agriculture Faisalabad, Pakistan. Treatments consisted of heat stress in main plots, viz. H₀ = no heat stress imposition, and H₁ = heat imposition for 14 d after emergence at seedling stage, and 15 cultivars of cotton in subplots, viz. FH-Lalazar, FH-142, FH-114, CIM-598, CIM-599, CIM-602, VH-282, VH-326, VH-341, MNH-886, MNH-888, MNH-992, IUB-13, IUB-212, and IUB-222. Decreases in relative leaf water contents and cell membrane thermo-stability were lower for genotypes CIM-598, CIM-599, CIM-602, VH-282, VH-326, VH-341, MNH-888, MNH-992 and IUB-13 compared to other cultivars. Antioxidant enzymes were enhanced under heat stress compared to the control for cultivars CIM-598, CIM-599, CIM-602, VH-282, VH-326, VH-341, MNH-888, MNH-992 and IUB-13, while these were decreased in all other cultivars. Heat-mediated decreases in root, shoot length, fresh and dry weight were lower for cultivars CIM-598, CIM-599, CIM-602, VH-282, VH-326, VH-341, MNH-888, MNH-992 and IUB-13 compared to other genotypes. Essentially, heat stress deleteriously impacted all the evaluated parameters; however, distinct cotton genotypes varied remarkably from each other for antioxidants, membrane stability and biomass accumulation. Moreover, biochemical markers proved to be potential regulators of biomass accumulation and hence can be used as road map in future cotton improvement programs. On the basis of the studied response variables, genotypes CIM-598, CIM-599, CIM-602, VH-282, VH-326, VH-341, MNH-888, MNH-992 and IUB-13 manifested heat tolerance, while FH-114 showed susceptibility to heat stress in cotton.