Epigenetics plays an important role in biological processes of microorganisms. DNA modification with restriction-modification system is a component of microbial immune system, and DNA modification without restriction system influences phenotypes by gene regulation. However, epigenetic information has not been collected for DNA sequence analysis routinely. Single molecule real time (SMRT) sequencing, based on analysis of DNA synthesis kinetics, enables the acquisition of DNA primary sequences and detection of modified nucleotides simultaneously. This technology provides a new platform for research on known DNA modifications and discovery of novel DNA modifications. This review summarized single molecule real time sequencing and its applications in microbial epigenetics.