IntroductionPediatric and neonatal sepsis is one of the main causes of mortality and morbidity in these age groups. Accurate and early etiological identification is essential for guiding antibiotic treatment, improving survival, and reducing complications and sequelae. Currently, the identification is based on culture-dependent methods, which has many limitations for its use in clinical practice, and obtaining its results is delayed. Next-generation sequencing enables rapid, accurate, and unbiased identification of multiple microorganisms in biological samples at the same time. The objective of this study was to characterize the etiology of neonatal and pediatric sepsis by metagenomic techniques.MethodsA systematic review of the literature was carried out using the PRISMA-2020 guide. Observational, descriptive, and case report studies on pediatric patients were included, with a diagnostic evaluation by clinical criteria of sepsis based on the systemic inflammatory response, in sterile and non-sterile biofluid samples. The risk of bias assessment of the observational studies was carried out with the STROBE-metagenomics instrument and the CARE checklist for case reports.Results and DiscussionFive studies with a total of 462 patients were included. Due to the data obtained from the studies, it was not possible to perform a quantitative synthesis (meta-analysis). Based on the data from the included studies, the result identified that mNGS improves the etiological identification in neonatal and pediatric sepsis, especially in the context of negative cultures and in the identification of unusual microorganisms (bacteria that are difficult to grow in culture, viruses, fungi, and parasites). The number of investigations is currently limited, and the studies are at high risk of bias. Further research using this technology would have the potential to improve the rational use of antibiotics.