Denaturing gradient gel electrophoresis (DGGE) is a commonly used molecular technique for rapid fingerprint analysis of microbial community composition, diversity, and dynamics. The method is rapid and affordable, allowing multiple samples to be processed simultaneously. This protocol provides a background to the theory and progress in DGGE techniques, and offers a detailed step-by-step procedure for laboratories employing DGGE for the analysis of bacterial populations from environmental samples. Potential sources of bias are highlighted in addition to a detailed troubleshooting section that helps to overcome common problems associated with DGGE analyses. The protocol outlines steps for preparing gel solutions, pouring gels, operating the DGGE apparatus, and excising fingerprint bands of interest for sequencing. All the required reagents and supplies are listed, as are the sequences of the most commonly used PCR primer sets for DGGE of bacterial 16S rRNA gene amplicons. A comparison of the three major providers of DGGE apparatuses is also included.