Understanding how microbial genotypes are arrayed in space is crucial for identifying local factors that may influence the spatial distribution of genetic diversity. In this study we investigated variation in 16S rDNA sequences and rep-PCR fingerprints of Streptomyces stains isolated from prairie soil among three locations and four soil depths. Substantial variation in Streptomyces OTU (operational taxonomic unit) and BOX-PCR fingerprint diversity was found among locations within a limited spatial area (1 m2). Further, phylogenetic lineages at each location were distinct. However, there was little variation in genetic diversity among isolates from different soil depths and similar phylogenetic lineages were found at each depth. Some clones were found at a localized scale while other clones had a relatively widespread distribution. There was poor correspondence between 16S rDNA groupings and rep-PCR fingerprint groupings. The finding of distinct phylogenetic lineages and the variation in spatial distribution of clones suggests that selection pressures may vary over the soil landscape.