Background <p>Infantile hydrocephalus is a challenging condition with variable timing of clinical recognition. Early identification of newborns at increased risk could enable closer surveillance and earlier intervention. Neonatal blood samples collected routinely for newborn screening allow us to test whether specific blood biomarkers are associated to hydrocephalus.</p> Methods <p>We used the data from an established nationwide Danish cohort study of 14,866 infants born from March 2009 to March 2011, with blood samples collected 2–8 days after birth and linked to longitudinal health registry data. The data included ten protein biomarkers spanning from inflammatory, cellular stress, growth, and neurotrophic pathways. Associations between neonatal biomarker concentrations and hydrocephalus were investigated using multivariable regression models adjusting for gestational age, age at sampling, and neonatal anthropometrics.</p> Results <p>Among 14,866 infants, 41 were diagnosed with hydrocephalus (approximately 3 per 1,000 births). The primary biomarker analyses included 30 hydrocephalus cases diagnosed no later than the age of 2 years and 14,577 controls. Neonatal heat-shock protein 70 (HSP70) concentrations were significantly higher in infants who developed hydrocephalus. No significant differences were found for the remaining biomarkers, and subgroup analyses related to intraventricular hemorrhage did not show distinct biomarker profiles.</p> Conclusions <p>In this nationwide cohort, elevated HSP70 in neonatal blood samples was associated with development of infantile hydrocephalus. No other of the inflammatory and neurotrophic biomarkers we investigated were significantly associated with hydrocephalus. These findings support further mechanistic investigation of HSP70 as a potential component of multifactorial early-life risk models of hydrocephalus.</p>