The purpose of this prospective ultrasound-based pilot study was to identify the most suitable tibial puncture site for intraosseous (IO) access in term and preterm neonates, describe tibial dimensions at this site, and provide anatomical landmarks for rapid localization. We measured the tibial dimensions and distances to anatomical landmarks at puncture sites A (proximal: 10 mm distal to the tibial tuberosity; distal: 10 mm proximal to the malleolus medialis) and B (chosen by palpation of the pediatrician), in 40 newborns in four weight groups (< 1000 g; 1000–2000 g, 2000–3000 g, and 3000–4000 g). Sites were rejected if they fell short of the assumed safety distance to the tibial growth plate of 10 mm. If both A and B were rejected, puncture site C was determined sonographically at the maximum tibial diameter while maintaining the safety distance. Puncture site A violated the safety distance in 53% and 85% (proximally and distally, respectively) and puncture site B in 38% and 33%. In newborns weighing 3000–4000 g, at median (IQR), the most suitable puncture site at the proximal tibia was 13.0 mm (12.0–15.8) distal to the tuberosity and 6.0 mm (4.0–8.0) medial to the anterior rim of the tibia. The median (IQR) diameters at this site were 8.3 mm (7.9–9.1) (transverse) and 9.2 mm (8.9–9.8) (anterior–posterior). The diameters increased significantly with increasing weight. Conclusion: This study adds concise, practical information on the implementation of IO access in neonatal patients: the tibial dimensions in newborns in four different weight groups and initial data on anatomical landmarks to easily locate the IO puncture site. The results may help implement IO access in newborns more safely.What is Known:• Intraosseous access is a feasible option for emergency administration of vital drugs and fluids in newborns undergoing resuscitation when an umbilical venous catheter is impossible to place.• Severe complications of IO access due to malpositioned IO needles have been reported in neonates.What is New:• This study reports the most suitable tibial puncture sites for IO access and the tibial dimensions, in newborns of four weight groups.• The results can help to implement safe IO access in newborns.