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The aim of this article is to discuss regional impacts of high-speed railways on transportation accessibility. It investigates the potential changes in the transportation accessibility of Czech municipalities due to the planned construction of high-speed rail lines in Czechia. It considers two currently discussed variants of the high-speed rail line between Prague and Brno. We discuss the model of potential transportation accessibility and discuss its usability in the preparation and construction of high-speed rail line structures, and how it would affect the optimal route selection. We also present different methods of accessibility analyses in GIS applied to the case of Czechia and their presentation through map outputs.