Background: Despite iron supplementation, some preterm infants develop iron deficiency (ID). The optimal iron status parameter for early detection of ID has yet to be determined. Objective: To establish reference ranges for reticulocyte haemoglobin content (Ret-He) in preterm and term infants and to identify confounding factors. Methods: Retrospective analyses of Ret-He and complete blood count in infants with a clinically indicated blood sample obtained within 24 h after birth. Results: Mean (SD) Ret-He was 30.7 (3.0) pg in very preterm infants with a gestational age (GA) of <30 weeks (n = 55), 31.2 (2.6) pg in moderately preterm infants (GA 30-36 weeks, n = 241) and 32.0 (3.2) pg in term infants (GA ≥37 weeks, n = 216). The 2.5th percentile of Ret-He across all GA groups was 25 pg, with a weak correlation between Ret-He and GA (r = 0.18). Moreover, only weak/no correlations were found between Ret-He and C-reactive protein (r = 0.18), interleukin 6 (IL-6) (r = 0.03) and umbilical artery pH (r = -0.07). There was a slight variation in Ret-He with mode of delivery [normal vaginal delivery: 32.3 (3.2) pg, secondary caesarean section (CS): 31.4 (3.0) pg, instrumental delivery: 31.3 (2.7) pg and elective CS: 31.2 (2.8) pg]. Conclusion: GA at birth has a negligible impact on Ret-He, and the lower limit of the normal reference range in newborns within 24 h after birth can be set to 25 pg. Moreover, Ret-He seems to be a robust parameter which is not influenced by perinatal factors within the first 24 h after birth.