Polyphemus pediculus is a freshwater predatory crustacean with a highly specialized compound eye adapted for visually guided behaviour. Although structural regionalization within the eye has previously been described, the spatial localization of opsin gene expression in this species remains poorly understood. This study investigated the localization of selected opsin genes in the eye of P. pediculus using RNA in situ hybridization while also evaluating fixation and cryosectioning conditions for improved tissue preservation during hybridization procedures. Selected opsin genes were amplified, sequenced and used for synthesis of DIG-labelled antisense RNA riboprobes. Different fixation and cryosectioning approaches were tested prior to in situ hybridization. Cryosections prepared on the same day following fixation showed better tissue preservation and partial loss of sections during hybridization and washing procedures. Previously stored cryosections retained good tissue morphology when same day cryosectioning had been performed, indicating that storage itself was not the primary cause of tissue damage. Although non-specific background staining remained a limitation, localized staining patterns were observed in some retinal regions following hybridization with selected opsin riboprobes. Overall, the study highlights the importance of optimized fixation and cryosectioning conditions for successful RNA in situ hybridization in P. pediculus and provides a basis for future studies investigating visual organization and opsin expression in freshwater crustaceans.