Abstract Spatial and temporal variability of soil bacterial 16S rDNA terminal restriction fragment (TRF) size variation was evaluated in a homogeneous grassland (HG) dominated by the turf-forming grass Bouteloua gracilis and in a shrubland (SL) dominated by Artemisia tridentata (Wyoming big sagebrush). Temporal variability was also evaluated on the HG site over a growing season. No trends toward dissimilarity were detected with temporal (180 days) or spatial (up to 100 m) distance in the HG system. Terminal-restriction fragment length polymorphism (T-RFLP) profiles of the SL site exhibited pronounced small-scale spatial variability (<70 cm), although spatial analysis indicated weak spatial autocorrelation to distances greater than 36 cm. While shrub-induced nutrient localization was shown to significantly influence T-RFLP profiles, very little of the variability could be accounted for on the basis of spatial characteristics, suggesting that soil bacterial 16S rDNA composition of this site is predominantly controlled at scales other than those measured. Average dissimilarity values differed greatly between the two sites (0.27 and 0.59 for HG and SL sites, respectively). These results suggest that plant community structure strongly influences bacterial community composition in these semiarid ecosystems, highlighting the importance of considering spatial variability when designing field studies related to bacterial diversity in ecosystems having patchy or heterogeneous plant cover.