Terminal restriction fragment length polymorphism (T‐RFLP) analysis is a polymerase chain reaction (PCR)‐fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR‐fingerprinting methods. Sizing of terminal restriction fragments (T‐RFs) can now be done using capillary sequencing technology allowing samples contained in 96‐ or 384‐well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T‐RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T‐RFLP data. Due to biases inherent in the method, linking the size of T‐RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T‐RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T‐RFLP method is a user‐friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.