The article reviews the application of next-generation sequencing (NGS) in identifying different pathogens, including bacteria, fungi, and viruses. It highlights the advancements in sequencing technologies, from Sanger sequencing to NGS and third-generation sequencing (3GS). Sanger sequencing, while still used for small-scale sequencing, has been surpassed by NGS due to its high-throughput and cost-effectiveness. NGS has revolutionized pathogen identification by enabling the simultaneous sequencing of millions of DNA fragments, providing detailed genetic information about pathogens. The article discusses the advantages of NGS, such as its ability to detect unknown pathogens, identify virulence factors, and track resistance mechanisms. It also covers specific applications of NGS in whole-genome sequencing (WGS), targeted next-generation sequencing (tNGS), and metagenomic next-generation sequencing (mNGS). WGS is particularly useful for identifying unknown organisms and tracking the spread of bacterial pathogens. tNGS is effective for amplifying and sequencing specific gene fragments, while mNGS can detect all genetic material in a sample. The article concludes by emphasizing the potential of NGS in improving the diagnosis, surveillance, and control of infectious diseases, despite challenges such as the need for robust bioinformatics pipelines and high diagnostic costs.The article reviews the application of next-generation sequencing (NGS) in identifying different pathogens, including bacteria, fungi, and viruses. It highlights the advancements in sequencing technologies, from Sanger sequencing to NGS and third-generation sequencing (3GS). Sanger sequencing, while still used for small-scale sequencing, has been surpassed by NGS due to its high-throughput and cost-effectiveness. NGS has revolutionized pathogen identification by enabling the simultaneous sequencing of millions of DNA fragments, providing detailed genetic information about pathogens. The article discusses the advantages of NGS, such as its ability to detect unknown pathogens, identify virulence factors, and track resistance mechanisms. It also covers specific applications of NGS in whole-genome sequencing (WGS), targeted next-generation sequencing (tNGS), and metagenomic next-generation sequencing (mNGS). WGS is particularly useful for identifying unknown organisms and tracking the spread of bacterial pathogens. tNGS is effective for amplifying and sequencing specific gene fragments, while mNGS can detect all genetic material in a sample. The article concludes by emphasizing the potential of NGS in improving the diagnosis, surveillance, and control of infectious diseases, despite challenges such as the need for robust bioinformatics pipelines and high diagnostic costs.