Heat stress poses a major threat to plant productivity and crop yields. The induction of heat shock proteins (HSPs) by heat shock factors is a principal defense response of plants exposed to heat stress. Here, we identified and analyzed the heat stress-induced Whirly1 (SlWHY1) gene in tomato (Solanum lycopersicum). We generated various SlWHY1-overexpressing (OE) and SlWHY1-RNAi lines to investigate the role of WHY1 in thermotolerance. Compared to the wild type (WT), the OE lines showed less wilting, as reflected by their increased membrane stability and soluble sugar content and reduced reactive oxygen species (ROS) accumulation under heat stress. By contrast, RNA interference (RNAi) lines with inhibited SlWHY1 expression showed the opposite phenotype and corresponding physiological indices under heat stress. The heat-induced gene SlHSP21.5A, encoding an endoplasmic reticulum-localized HSP, was upregulated in the OE lines and downregulated in the RNAi lines compared to WT. RNAi-mediated inhibition of SlHSP21.5A expression also resulted in reduced membrane stability and soluble sugar content and increased ROS accumulation compared to the WT under heat stress. SlWHY1 binds to the elicitor response element-like element in the promoter of SlHSP21.5A to activate its transcription. These findings suggest that SlWHY1 promotes thermotolerance in tomato by regulating SlHSP21.5A expression.,© The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.