Abstract. Background: In infants with suspected neonatal sepsis, standard culture methods may fail to detect bacteremia due to a lower concentration of pathogens in the bloodstream. We sought to determine if MassTag PCR could accurately and reliably detect pathogens in neonates with a high suspicion of sepsis. Methods: Infants in a level III neonatal intensive care unit (NICU) were identiﬁed by the primary medical team as having clinical signs consistent with a high likelihood of infection. Samples of sterile bodily ﬂuid (i.e., blood and CSF) were obtained and evaluated both by standard culture methods and the study method. The study samples were analyzed by MassTag-PCR using primers that targeted twelve common neonatal bacterial pathogens. Results: Samples were collected from a total of 32 infants (gestational age 24-40 weeks) with clinical sepsis. There were eight infants with a positive result on MassTag PCR. One infant had Staphylococcus aureus identiﬁed in the blood by MassTag PCR, in whom the culture was also positive. In addition, there was one infant with a positive MassTag assay for S. aureus in the CSF. This infant had a positive blood culture for S. aureus and negative CSF culture, with normal cell counts and chemistry values. There were ﬁve infants with negative blood cultures, but a positive result on MassTag PCR for Pseudomonasthree, Enterococcus one, and Propionibacterium acnes one. There was one PCR assay considered to be a contaminant with Staphylococcus species. Conclusions: MassTag PCR could potentially serve as an important method of pathogen detection in the NICU, in particular for gram-negative and anaerobic pathogens. Further studies should investigate whether infants with positive MassTag PCR and negative culture truly represent infections that could not be detected by routine blood culture.