Heatwaves resulting from global warming are a leading threat to cotton crops because they specifically affect reproductive (gametophytic) development. We investigated pollen development using proteomics after exposure to heat in order to identify how gene products respond in tetrads, uninucleate and binucleate microspores, 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 heat stress resulted in differential expression of 170 – 880 proteins at different stages of development. Differentially expressed proteins were associated with structural molecule activity, transporter activity, multicellular organismal process, amino acid activation and cell cycle. We conclude that protein abundances responded differentially to heat according to when it was applied, for example by supporting cell division and growth in the haploid germ line. Physiological and proteomic evidence supported our observation that the sensitivity of pollen to heat declined as the gametophyte matured. Identifying the stage-specific proteins will reveal key heat-responsive genes and new genetic tools for improved resilience of crops as climates keep warming.