Semi-natural grasslands are a type of grassland that can develop independently of climatic suitability. In recent years, it has become increasingly recognized that grasslands maintained in close association with human livelihoods over long periods of time—often for centuries or more—form plant communities that differ entirely from the surrounding vegetation. In Japan, where such grasslands are distributed across the archipelago, these habitats are thought to have served as modern refugia for grassland species that dispersed from the continent during the Pleistocene. Indeed, many continental species that were once widespread are now found in Japan as isolated and declining populations. At the same time, numerous endemic species that have diversified more recently are also well documented within these grasslands. This study evaluates the patterns and processes of biodiversity gradients in long-persisting semi-natural grasslands from a previously missing mesoscale (thousands-of-kilometers) perspective. Specifically, we examine the structure of alpha- and beta-diversity referring to the natural grasslands and those with shorter persistence, connecting them with historical range dynamics under post-glacial climate change and changing anthropogenic influences.