Abstract: With the increasing importance of the prevention and treatment of infectious diseases in health care and the increasing social concern caused by infectious diseases, new technologies are crucial for preventing and controlling the spread of pathogens. The microbiology laboratory has always being playing a significant role as the first line of pathogen detection in infection control by carrying on smear microscopy, culture, identification, and susceptibility testing. Information provided by traditional molecular diagnostics and genotyping methods is limited, which can not satisfy the requirement of epidemiologic investigation on the outbreak and spread of communicable diseases. Next-generation sequencing determines the DNA sequence of a complete bacterial genome in a single sequencing run, from which information on resistance, virulence and typing is obtained. It is useful for investigation into the outbreak. The obtained genome data can be further used for developing an outbreak-specific screening test. In this review, a general introduction to next-generation sequencing and its applications in clinical microbiology including outbreak management, identification of unknown pathogens, taxonomy, and research on resistant genomes are presented.