BACKGROUND: Human parechovirus 3 (HPeV-3) and enteroviruses (EV) are commonly detected viruses in febrile neonates and young infants and are usually diagnosed by PCR. However, in this population, data on detection rates for samples from different anatomical sites are limited. OBJECTIVES: To determine PCR detection rates for HPeV-3 and EVs in serum and cerebrospinal fluid (CSF) samples from febrile neonates and young infants. STUDY DESIGN: This prospective study identified viruses in serum and CSF samples collected from febrile neonates and young infants (age <4 months) in Niigata, Japan, during 2014-2018. HPeV-3 or EV infection was defined as a positive quantitative real-time PCR result for the virus in serum or CSF. Genotypes were identified by sequence analyses of the viral protein 1 region. RESULTS: Among 216 patients, we identified 56 HPeV-3-infected (26 %) and 48 EV-infected patients (22 %). All (56/56; 100 %) HPeV-3-infected patients had a positive PCR result for serum, and 49/56 (88 %) had a positive result for CSF. In EV-infected patients, 40/48 (83 %) were positive for serum, and 34/48 (71 %) were positive for CSF, and 22/48 (46 %) were positive for serum (n = 14) or CSF (n = 8). If only a CSF sample had been obtained, 7 (12 %) HPeV-3 infections and 14 (29 %) EV infections would have been undiagnosed. Detection rates in serum and CSF differed by genotype in EV-infected patients. CONCLUSIONS: Viral RNA detection rates differed between serum and CSF in HPeV-3- and EV-infected neonates/infants. Combined evaluation of serum and CSF samples is important for accurate viral diagnosis in this population.