Background: Conventional methods of screening for Hirschsprung disease (HD) in newborns (barium enema, BE; anorectal manometry, ARM; rectal suction biopsy, RSB) have limitations and/or are invasive. High-resolution anorectal manometry (HR-ARM) is a minimally invasive technique that has potential to overcome most of these limitations, but normative data and performance characteristics have not been reported in newborns. The aims of our study were to assess anorectal sphincter metrics including resting pressure (RP), anal canal length (ACL), and rectoanal inhibitory reflex (RAIR) in healthy and asymptomatic newborns, and to explore the role of HR-ARM in the diagnosis of HD using these normal parameters. Methods: All procedures were performed using solid state HR-ARM equipment (Medical Measurement Systems, Enchede, The Netherland) by a single operator. In the first phase, 180 asymptomatic newborns (term newborns 95, preterm newborns 85) were studied, and anal RP, ACL, and RAIR were measured. In the second phase, 16 newborns with clinical manifestations of HD were studied (9 of whom had histopathologic confirmation), and parameters compared to asymptomatic newborns. Key Results: Normative RP values were higher in term newborns compared with preterm newborns (p < 0.05), and correlated with age. Progressive maturation of the anal sphincter was evident with chronologic age, both in preterm and term newborns. RAIR was present in all normal subjects. Using absent RAIR as indicative of HD, HR-ARM had a sensitivity 89% and specificity of 83% compared to RSB; these performance characteristics were better than BE (sensitivity 78%, specificity 17%), with significantly higher diagnostic accuracy (80% vs 53%, respectively, p = 0.009). Conclusions & Inferences: Anorectal sphincter pressure progressively matures with incremental increase in RP during the first months of life. HR-ARM is an effective and safe method that complements the diagnosis of HD in newborns. © 2014 John Wiley & Sons Ltd.