DNA profiling of microbial communities has been proposed as a tool for forensic comparison of soils, but its potential to discriminate between soils from similar land use and/or geographic location has been largely unexplored. We tested the ability of terminal restriction fragment length polymorphism (T-RFLP) to discriminate between soils from 10 sites within the Greater Wellington region, New Zealand, based on their bacterial and fungal DNA profiles. Significant differences in bacterial and fungal communities between soils collected from all but one pair of sites were demonstrated. In some instances, specific terminal restriction fragments were associated with particular sites. Patch discrimination was evident within several sites, which could prove useful for site-specific matching (e.g., matching shoe/car tire print to an object). These results support the need for further understanding of the spatial distribution of soil microbial communities before DNA profiling of soil microbial communities can be applied to the forensic context.