DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.