Abstract: The widespread prevalence of multidrug-resistant organisms in clinical settings poses significant challenges to the prevention and control of hospital-associatal infections. Novel gene sequencing techniques, such as whole-genome sequencing (WGS) and metagenomic next-generation sequencing (mNGS), have emerged as revolutionary tools for precisely tracing to the source of hospital-associatal infection outbreak and the prevention and control through high-resolution genomic analysis. The technical principles and advantages of WGS and mNGS were systematically reviewed in the article. The pivotal roles of the techniques in confirmation of outbreak, identification of infection source, transmission chain rebuilding, study on transmission dynamics and evaluation of effect on infection prevention and control were elaborated through analysis of typical cases in China and abroad so as to provide theoretical bases and technical support for precise identification of prevention and control of nosocomial infection.