Inducible promoters provide remarkable utility when sustained transgene expression compromises plant development or agronomic performance. In this study, four different plant heat shock protein (HSP) promoters were characterized in the vegetative tissues of stably transformed sugarcane to evaluate their efficacy and spatial expression profiles when directing the expression of a uidA reporter gene. This study produced new quantitative knowledge on the temporal and spatial expression of HSP promoters in sugarcane, expanding the promoter toolbox for crop biotechnology to support gene function studies and biotechnology applications, including precision gene editing and complex metabolic engineering.