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Abstract PremiseDetecting clear tissue‐ and organ‐specific patterns of gene expression is key to understanding the genetic mechanisms that control plant development. In situ hybridization (ISH) of mRNA is one of the most precise, yet most challenging approaches to gene expression assays. Methods and ResultsDetection of histone H4 expression in reproductive tissues ofMimulus lewisii, a model angiosperm, was optimized using the RNAscope ISH assay. The optimized protocol was used to detect histone H4 expression in reproductive tissues of two gymnosperm species,Taxodium distichumandJuniperus virginiana, without further need for species‐specific optimization. Additionally, the optimized protocol was used to detect expression ofCYCLOIDEAtranscription factors inM. lewisiireproductive tissues without further optimization and with results similar to those previously reported. ConclusionsThe RNAscope assay can quickly and sensitively generate high‐quality ISH results in reproductive tissues across a breadth of plant species.