The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using hybridization and other molecular approaches. In their usual format, such assays are based on the presence of unique subsequences in the target RNA and require a prior knowledge of what organisms are likely to be in a sample. They are thus limited in generality when analyzing an unknown sample.Herein, we demonstrate the utility of catalogs of masses to characterize the bacterial 16S rRNA(s) in any sample. Sample nucleic acids are digested with a nuclease of known specificity and the products characterized using mass spectrometry. The resulting catalogs of masses can subsequently be compared to the masses known to occur in previously-sequenced 16S rRNAs allowing organism identification. Alternatively, if the organism is not in the existing database, it will still be possible to determine its genetic affinity relative to the known organisms.