For many years, sequencing of the 16S ribosomal RNA (rRNA) gene has served as an important tool for determining phylogenetic relationships between bacteria. The features of this molecular target that make it a useful phylogenetic tool also make it useful for bacterial detection and identification in the clinical laboratory. Sequence analysis of the 16S rRNA gene is a powerful mechanism for identifying new pathogens in patients with suspected bacterial disease, and more recently this technology is being applied in the clinical laboratory for routine identification of bacterial isolates. Several studies have shown that sequence identification is useful for slow-growing, unusual, and fastidious bacteria as well as for bacteria that are poorly differentiated by conventional methods. The technical resources necessary for sequence identification are significant. This method requires reagents and instrumentation for amplification and sequencing, a database of known sequences, and software for sequence editing and database comparison. Commercial reagents are available, and laboratory-developed assays for amplification and sequencing have been reported. Likewise, there are an increasing number of commercial and public databases. Despite the availability of resources, sequence-based identification is still relatively expensive. The cost is significantly reduced only by the introduction of more automated methods. As the cost decreases, this technology is likely to be more widely applied in the clinical setting.