The use of biota to analyze the spatial range and distribution of biogeographic regions is essential to gain a better understanding of the ecological processes that cause biotic differentiation and biodiversity at multiple spatiotemporal scales. Recently, the collection of high-resolution biological distribution data (e.g., specimens) and advances in analytical theory have led to their quantitative analysis and more refined spatial delineation. This study was conducted to redefine floristic zones in the southern part of the Korean Peninsula and to better understand the eco-evolutionary significance of the spatial distribution patterns. Based on the distribution data of 309,333 vascular plant species in the Korean Peninsula, we derived floristic zones using self-organizing maps. We compared the characteristics of the derived regions with those of historical floristic zones and ecologically important environmental factors (climate, geology, and geography). In a clustering analysis of the floristic assemblages, four distinct regions were identified, namely, the cold floristic zone (Zone I) in high-altitude regions at the center of the Korean Peninsula, cool floristic zone (Zone II) in high-altitude regions in the south of the Korean Peninsula, warm floristic zone (Zone III) in low-altitude regions in the central and southern parts of the Korean Peninsula, and maritime warm floristic zone (Zone IV) including the volcanic islands of Jejudo and Ulleungdo. A total of 1,099 taxa were common to the four floristic zones. Zone IV had the highest abundance of specific plants (those found in only one zone), with 404 taxa. This study improves floristic zone definitions using high-resolution regional biological distribution data. It will help better understand and re-establish regional species diversity. In addition, our study provides key data for hotspot analysis techniques required for the conservation of plant diversity.