The general incidence of copper (Cu), zinc (Zn), and boron (B) deficiencies in soils of the Canadian prairies may be related to identifiable, highly variable, inherent soil attributes. The objective of this study was to investigate the variability of selected properties and their relationship with the bioavailability, forms, and distribution of Cu, Zn, and B in a range of prairie soils. The nature of these micronutrient distributions were evaluated by measuring extractable concentrations, supply rates, and by separation into various chemical pools through sequential extraction and spectroscopic speciation analyses. Soil pH was found to be the least variable property [coefficient of variation (CV) < 13%], whereas carbonate content was the most variable (CV > 130%). The Cu and B availability showed strong negative correlation with the sand content in all soils. Path coefficient results indicated that organic carbon had the highest positive direct effect on availability and supply of Cu and B in Grey soils. Extractable Zn was positively correlated with organic carbon content of Brown and Dark Brown soils. Overall, high sand content and low organic matter were identified as important soil properties contributing to the deficiency of Cu, Zn, and B. The major proportion of Cu, Zn, and B was found in the recalcitrant residual fraction (59%–88%), with the smallest proportions in labile soluble, exchangeable forms (2%–8%). The X-ray absorption near edge structure revealed that Cu and Zn associated with carbonate minerals were dominant forms of these micronutrients present in all soils. Chemisorption is likely a major process regulating the bioavailability of Cu and Zn in prairie soils.