BACKGROUND: Although neonatal herpes simplex virus (HSV) is a potentially devastating infection requiring prompt evaluation and treatment, large-scale assessments of the frequency in potentially infected infants have not been performed. METHODS: We performed a retrospective cross-sectional study of infants .60 days old who had cerebrospinal fluid culture testing performed in 1 of 23 participating North American emergency departments. HSV infection was defined by a positive HSV polymerase chain reaction or viral culture. The primary outcome was the proportion of encounters in which HSV infection was identified. Secondary outcomes included frequency of central nervous system (CNS) and disseminated HSV, and HSV testing and treatment patterns. RESULTS: Of 26 533 eligible encounters, 112 infants had HSV identified (0.42%, 95% confidence interval [CI]: 0.35%.0.51%). Of these, 90 (80.4%) occurred in weeks 1 to 4, 10 (8.9%) in weeks 5 to 6, and 12 (10.7%) in weeks 7 to 9. The median age of HSV-infected infants was 14 days (interquartile range: 9.24 days). HSV infection was more common in 0 to 28-day-old infants compared with 29- to 60-day-old infants (odds ratio 3.9; 95% CI: 184.108.40.206). Sixty-eight (0.26%, 95% CI: 0.21%.0.33%) had CNS or disseminated HSV. The proportion of infants tested for HSV (35%; range 14%.72%) and to whom acyclovir was administered (23%; range 4%.53%) varied widely across sites. CONCLUSIONS: An HSV infection was uncommon in young infants evaluated for CNS infection, particularly in the second month of life. Evidence-based approaches to the evaluation for HSV in young infants are needed.