Managing plant biodiversity in tallgrass prairie ecosystems under changing environmental conditions requires a better understanding of the mechanisms that control and maintain species' distributions. In a landscape with a clear spatial gradient of soil moisture, the concept of "hydrological niche" segregation states that the partitioning of water availability across space significantly explains the pattern of plant community composition and distribution. We evaluated the applicability of this concept to a tallgrass prairie remnant (the Faville Prairie in southern Wisconsin) with a 70-year monitoring history, the Faville Prairie Wisconsin State Natural Area (43.1486N, 88.8779W). We tested the hypothesis that plants occupy distinct hydrological niches by partitioning the soil moisture availability gradient generated by topographic heterogeneity. Using a permanent array of 180 plots across the prairie, we collected plant occurrences in 2022 and topsoil moisture contents monthly from May to August in 2023. We made continuous soil moisture maps with spatial interpolation in ArcGIS Pro. Our results suggest that fine-grained topographic heterogeneity generates significant spatial and temporal variation in soil moisture at Faville. The distribution of plant species differed significantly along the spatial gradient of soil moisture. We describe these species-specific patterns of segregation and posit explanations for each. These results support the conclusion that plants at Faville coexist at a landscape level by segregating based on distinct hydrological niches. This study provides more insight into understanding the mechanisms that govern plant distribution in a tallgrass prairie by taking hydrological affinities of species into account.