From 2001 to 2023, 17 (14%) of 120 neonates with confirmed herpes simplex virus infection tested positive for herpes simplex virus by polymerase chain reaction (PCR) from only mucosal sites without a clinical mucosal lesion. Whether mucosal PCR positivity reflects early infection that may lead to recognizable disease, transient colonization, or a false-positive PCR result remains a clinical conundrum and warrants further study.