Heat stress induces severe meiotic defects in plants, leading to significant male sterility and substantial yield losses in crops. However, few genes involved in meiotic thermotolerance have been characterized. In this study, we demonstrate that HSP101, a conserved heat shock protein, plays a critical role in protecting pollen mother cells from heat-induced meiotic defects in both rice and Arabidopsis. HSP101 is highly expressed during early pollen development, and its loss of function leads to meiotic instability under heat stress. Transcriptomic analysis revealed that HSP101 deficiency disrupts the transcriptional network essential for cellular homeostasis during heat stress. Importantly, overexpression of HSP101 enhanced thermotolerance during microsporogenesis without obvious adverse effects on plant growth. Our findings establish HSP101 as a positive regulator of meiotic thermotolerance during microsporogenesis using rice and Arabidopsis as model systems, providing critical insights for improving adaptation of male meiocytes to high temperatures in crops.