Background: Neonatal Intensive Care Unit (NICU) patients are at increased risk for autism spectrum disorder (ASD). Autonomic nervous system aberrancy has been described in children with ASD, and we aimed to identify heart rate (HR) patterns in NICU patients associated with eventual ASD diagnosis. Methods: This retrospective cohort study included NICU patients from 2009-2016 with archived HR data and follow-up beyond age 3 years. Medical records provided clinical variables and ASD diagnosis. HR data were compared in infants with and without ASD. Results: Of 2371 patients, 88 had ASD, and 689,016 hours of data were analyzed. HR skewness (HRskw) was significantly different between ASD and control infants. Preterm infants at early postmenstrual ages (PMA) had negative HRskw reflecting decelerations, which increased with maturation. From 34-42 weeks PMA, positive HRskw toward accelerations was higher in males with ASD. In 931 males with at least 4 days of HR data, overall ASD prevalence was 5%, whereas 11% in the top 5 th HRskw percentile had ASD. Conclusion: High HRskw in NICU males, perhaps representing autonomic imbalance, was associated with increased ASD risk. Further study is needed to determine whether HR analysis identifies highest risk infants who might benefit from earlier screening and therapies.