The ever-changing global environment currently includes an increasing ambient temperature that can be a devastating stress for organisms. Plants, being sessile, are adversely affected by heat stress in their physiology, development, growth, and ultimately yield. Since little is known about the response of biochemical traits to high-temperature ambiance, we evaluated eight parental lines (five lines and three testers) and their 15 F hybrids under normal and high-temperature stress to assess the impact of these conditions over 2 consecutive years. The research was performed under a triplicate randomized complete block design including a split-plot arrangement. Data were recorded for agronomic, biochemical, and fiber quality traits. Mean values of agronomic traits were significantly reduced under heat stress conditions, while hydrogen peroxide, peroxidase, total soluble protein, superoxide dismutase, catalase (CAT), carotenoids, and fiber strength displayed higher mean values under heat stress conditions. Under both conditions, high genetic advance and high heritability were observed for seed cotton yield (SCY), CAT, micronaire value, plant height, and chlorophyll-a and b content, indicating that an additive type of gene action controls these traits under both the conditions. For more insights into variation, Pearson correlation analysis and principal component analysis (PCA) were performed. Significant positive associations were observed among agronomic, biochemical, and fiber quality-related traits. The multivariate analyses involving hierarchical clustering and PCA classified the 23 experimental genotypes into four groups under normal and high-temperature stress conditions. Under both conditions, the F hybrid genotype FB-SHAHEEN × JSQ WHITE GOLD followed by Ghuari-1, CCRI-24, Eagle-2 × FB-Falcon, Ghuari-1 × JSQ White Gold, and Eagle-2 exhibited better performance in response to high-temperature stress regarding the agronomic and fiber quality-related traits. The mentioned genotypes could be utilized in future cotton breeding programs to enhance heat tolerance and improve cotton yield and productivity through resistance to environmental stressors.