Abstract Background Climate change and particularly global warming has emerged as an alarming threat to the crop productivity of field crops and exerted drastic effects on the cropping patterns. Production of cotton has been dropped down to one million bales from 1.4 million bales since 2015 in Pakistan due to the increase in temperature at critical growth stages, i.e., flowering and boll formation. Keeping in view the importance of cotton in the country, this study was conducted to investigate the genetic effects conferring heat tolerance in six populations (P1, P2, F1, F2, BC1 and BC2) developed from cross-1 and cross-2, i.e., VH-282 × FH-142 and DNH-40 × VH-259. Results The results revealed that cross-1 performed better in heat stress as compared with cross-2 for majority of the traits recorded. Boll weight and ginning outturn (GOT) were highly effected under heat stress and had negative correlation with Relative cell injury (RCI). Boll weight, fiber length, fiber strength and fiber fineness were under the control of non-additive gene action, whereas RCI was controlled by additive gene effects. Lower values of genetic advance coupled with higher values of broad sense heritability for these traits except RCI confirmed the role of non-additive genetic effects. Duplicate types of epistasis were recorded for fiber strength in cross-1 in normal condition. However, complementary type of non-allelic interaction was recorded for fiber strength under normal condition, fiber fineness and RCI under heat stressed condition in cross-1. Likewise, boll weight, GOT and fiber length in populations derived from cross-2 in normal condition were also under the influence of complementary type of non-allelic interaction. Significant differences among values of mid parent and better parent heterosis for boll weight in both normal and heat stress condition provided the opportunity to cotton breeders for utilization of this germplasm for improvement of this trait through exploitation of heterosis breeding. Conclusion Cross-1 performed better in heat stress and could be utilized for development of heat tolerant cultivar. RCI was under the influence of additive gene action, so one can rely on this trait for screening of large number of accessions of cotton for heat stress. While other traits were predominantly controlled by non-additive gene action and selection based on these should be delayed in later generations.