Storage conditions are considered to be a critical component of DNA-based microbial community analysis methods. However, whether differences in short-term sample storage conditions impact the assessment of bacterial community composition and diversity requires systematic and quantitative assessment. Therefore, we used barcoded pyrosequencing of bacterial 16S rRNA genes to survey communities, harvested from a variety of habitats [soil, human gut (feces) and human skin] and subsequently stored at 20, 4, −20 and −80 °C for 3 and 14 days. Our results indicate that the phylogenetic structure and diversity of communities in individual samples were not significantly influenced by the storage temperature or the duration of storage. Likewise, the relative abundances of most taxa were largely unaffected by temperature even after 14 days of storage. Our results indicate that environmental factors and biases in molecular techniques likely confer greater amounts of variation to microbial communities than do differences in short-term storage conditions, including storage for up to 2 weeks at room temperature. These results suggest that many samples collected and stored under field conditions without refrigeration may be useful for microbial community analyses.