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Summary: Many tools have been developed to measure the degree of similarity between gene duplicates within and between species. Here, we present HSDecipher, a bioinformatics pipeline to assist users in the analysis and visualization of highly similar duplicate genes (HSDs). We describe the steps for analysis of HSDs statistics, expanding HSD gene sets, and visualizing the results of comparative genomic analyses. HSDecipher represents a useful tool for researchers exploring the evolution of duplicate genes in select eukaryotic species.For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021)1 and Zhang et al. (2022).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.