Ecological boundaries are critical landscape regions of transition between adjacent ecological systems. While environmental controls of boundaries may operate in a scale-dependent manner, multiple-scale comparisons of vegetation-environment relationships have been characterized for few boundary systems. We used approximately 250000 point records on the occurrence of woody versus grassland vegetation in conjunction with climatic, topographical, and soils data to evaluate scale effects and spatial heterogeneity in a 650-km section of the historic prairie-forest biome boundary of Minnesota, USA. We chose this as a model system because of the availability of historical vegetation data, a considerable spatial extent, a sharp ecological transition, and the ability to avoid confounding from more recent anthropogenic land use change. We developed modeling techniques using hierarchical variance partitioning in a spatially-structured format that allowed us to simultaneously evaluate vegetation-environment relationships across two-dimensional space (i.e. the prairie-forest boundary) and across spatial scales (i.e. varying extents). Soils variables displayed the least spatial autocorrelation at shortest lag distances and tended to be the least important predictors of woody vegetation at all spatial extents. Topographical variables displayed greater spatial heterogeneity in regions dominated by forest compared with prairie and were more important at fine-intermediate spatial scales, highlighting their likely control on fire regimes. An integrated climatic variable (precipitation minus potential evapotranspiration) displayed a trend of increasing spatial variance across the study region and was unambiguously the strongest biome boundary control, although its joint influence with fire was difficult to characterize. Spatially heterogeneous vegetation-environment relationships were observed at all scales, especially at finer scales. Our results suggest that the importance of environmental controls changes smoothly rather than discretely across scales and demonstrate the need to account for spatial non-stationarity and scale to predict and understand vegetation distribution across ecological boundaries.