Most soil microbial community studies to date have focused on homogenized bulk soil samples. However, it is likely that many important microbial processes occur in spatially segregated microenvironments in the soil leading to a microscale biogeography. This study attempts to localize specific microbial populations to different fractions or compartments within the soil matrix. Microbial populations associated with macroaggregates and inner- versus total-microaggregates of three diverse soils were characterized using culture-independent, molecular methods. Despite their relative paucity in most surveys of soil diversity, representatives of Gemmatimonadetes and Actinobacteria subdivision Rubrobacteridae were found to be highly abundant in inner-microaggregates of most soils analyzed. By contrast, clones affiliated with Acidobacteria were found to be relatively enriched in libraries derived from macroaggregate fractions of nearly all soils, but poorly represented in inner-microaggregate fractions. Based upon analysis of 16S rRNA, active community members within microaggregates of a Georgian Ultisol were comprised largely of Gemmatimonadetes and Rubrobacteridae, while within microaggregates of a Nebraska Mollisol, Rubrobacteridae and Alphaproteobacteria were the predominant active bacterial lineages. This work suggests that microaggregates represent a unique microenvironment that selects for specific microbial lineages across disparate soils.