High temperature negatively affects cotton production worldwide. In Pakistan, cotton crop faces high temperature at peak flowering during June–July, which is a major reason for yield losses. The present study was conducted to find some agro-physiological markers for high-temperature tolerance. Fifty cotton genotypes were raised under normal sown (high-temperature stress) and late sown (optimum temperature) conditions for 2 years. Data were recorded for relative cell injury percentage (RCI), chlorophyll content (CC), canopy temperature (CT), boll retention percentage (BR), bolls per plant (BP), boll weight (BW), hundred seed weight (HSW), sympodial branches per plant (SBP), plant height (PH) and seed cotton yield (SCY). A large amount of variability was found among cotton genotypes as proved by descriptive statistics. Stable genotypes were selected based on higher SCY and yield components (BP, BW, HSW, SBP, PH and SCY) performance along with less RCI%, high CC and low CT. The results of heatmap analysis showed that the six cotton genotypes viz., FH-Noor, FH-Lalazar, FH-458, FH-466, NIAB-545 and NIAB-878 performed better under high-temperature stress. These selected genotypes can be a source for breeding high-temperature tolerance. Furthermore, the results from correlation analysis confirmed that the traits such as RCI%, CC and CT can be considered as early selection criteria due to their positive association with SCY. Whilst some other parameters such as BP, BW, BR, SBP and PH might be used as direct selection indices for SCY under high-temperature stress.