Hotter climates threaten food security by adversely affecting crop fertility across the globe. Understanding the molecular responses of plants to heat will enable breading of resilient varieties suited to future climate regimes. In this study, cotton plants acclimated to 34°C for 1 d were exposed to 38/28°C (day/night) for 5 d. Vegetative (anthers and leaves) and reproductive (tetrads, uninucleate and binucleate microspores and mature pollen) tissues were collected and subjected to SWATH-MS proteomic analysis using a Triple TOF 6600 mass spectrometer (Sciex, USA) equipped with an Eksigent nanoLC 400 liquid chromatography system (Sciex, USA). We identified 4,501 proteins across all pollen developmental stages, 3,056 proteins in anthers, and 2,958 proteins in leaf tissues. Functional enrichment analysis of tetrads (the most heat-sensitive stage of pollen development) revealed high abundance of proteins associated with ribosomal processes after 5 d at 38°C. In contrast, ribosomal proteins generally decreased in abundance in mature pollen and leaves in response to high temperature, reflecting the suppression of translational processes in these more heat-tolerant tissues. Moreover, we found increased abundance of proteins related to protein transport in tetrads, whereas they were less common in later stages of pollen development and vegetative tissues. Identifying the precise molecular events that underlie the sensitivity of pollen formation to heat stress is now a top high priority for crop improvement.