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The fall of the Iron Curtain created a vacuum upon which large-scale collectivized agriculture was largely abandoned. Post-agricultural brownfields emerge in multiple manners across national, regional and local levels. While these sites remain rarely explored, we aimed to better understand the spatial consequences of the formation, persistence and reuse of these sites. The regions of South Bohemia and South Moravia in the Czech Republic are used to show the location of post-agricultural brownfields identified in 2004 through 2018. Using Global Moran’s I test we have found that post-agricultural brownfields existing in 2004, long-term brownfields in 2018 and brownfields established between 2004 and 2018 are spatially clustered, but remediated brownfields between 2004 and 2018 are not. Next, the Anselin’s Local Moran’s I test identified where the spatial clusters exist. The clusters identified were examined for differences in their social, economic and environmental development by the means of logistic regression. The results show that the brownfields initially identified in 2004 are concentrated in regions with lower quality agricultural land while simultaneously located in the hinterlands of regional urban centers. In contrast, peripheral regions most often contained long-term brownfields. Brownfield sites identified after 2004 occurred in regions with higher agricultural quality of land and where corn usually grows.