Spatial variation in soil properties is often considered significant across broad geographical regions due to soil formation factors. However, fine-scale variations might also be significant. This study was conducted with the original intent of assessing how simple and complex mixtures of annual forages might be used to renovate perennial pastures. Private farmers in the Flatwoods, Piedmont, and Blue Ridge Major Land Resource Areas of North Carolina tested annual forages to renovate tall fescue pastures. Soil was sampled in multiple random locations in each field at depths of 0-6, 6-12, and 12-20 cm at the beginning and ending of a 3-year annual forage evaluation. Relative variation among five components [year of sampling (n = 2), physiographic region (n = 3), annual forage treatment (n = 2), soil depth (n = 3), and random variation from pseudoreplicates (n = 3 in 2015 and n = 5 in 2018)] was assessed for four soil physical, 10 soil biological, and 16 soil chemical properties. Soil chemical properties were mostly affected by physiographic region (47 ± 26% of total variation) and soil depth (33 ± 18%), soil biological properties were mostly affected by soil depth (63 ± 25%) and random pseudoreplication (14 ± 6%), and soil physical properties were equally affected by pseudoreplication (35 ± 21%), physiographic region (32 ± 18%), and soil depth (29 ± 22%). The type of annual forage had no discernible effect on soil properties, even the most biologically active. A diversity of spatial variations was important, suggesting that regional-level ecological investigations require careful attention to an appropriate sampling design considering multiple factors. This article is protected by copyright. All rights reserved