Updated publication reference for PubMed record(s): 34837283. Heatwaves resulting from global warming are a leading threat to plants because they specifically impair reproductive (gametophytic) development. We investigated pollen development using proteomics after exposure to heat (38/28 °C; day/night). Sampling enabled the identification of the impact of heat on gene products in tetrads (immediately post-meiosis), uninucleate and binucleate microspores (early mitosis), and mature pollen. A library consisting of 5257 G. hirsutum proteins was constructed using SWATH-MS, which led to quantification of 4501 proteins at the four distinct stages. Data analysis revealed that 880, 360, 307 and 166 proteins were differentially expressed when the tetrad, uninucleate, binucleate and mature pollen stages, respectively, were subjected to heat. Surprisingly, most of these DEPs were identified only at a single developmental stage, indicating translation of stage-specific proteins in response to heat. Especially, high numbers of differentially expressed proteins (DEPs) were identified in tetrads compared with the late developmental stages; these were mainly associated with protein processing in endoplasmic reticulum, oxidative phosphorylation, spliceosome, cytoskeletal response, chaperones and folding catalysts, carbon metabolism and membrane trafficking. We conclude that protein synthesis in tetrad cells responded acutely to heat by supplying cellular components that enable subsequent mitosis and structural changes in the haploid germ cells leading the mature pollen. We hypothesise that fewer DEPs at the later stages might reflect a lower sensitivity to high temperatures. Identifying the stage-specific proteins will lead to the identity of key heat-responsive genes and new genetic tools for improved resilience of all seed and fruit-based crops as climates keep warming.