The spine of L4 usually lies on a line drawn between the highest points of the iliac crests (Tuffier's line) in adults. Although its accuracy has been questioned, it is still commonly used to identify the spinous process of the 4th lumbar vertebra before performing lumbar neuraxial procedures. In children, this line is said to cross the midline at the level of L5. A literature search revealed that the description this surface anatomical line is vague in neonates. The aims of this study were to determine the vertebral level of Tuffier's line, as well as its distance from the apex of the sacrococcygeal membrane (ASM), in 39 neonatal cadavers in both a prone and flexed position. It was found that when flexed, Tuffier's line shifted from the level of L4/L5 (prone position) to the upper third of L5. The mean distance from the ASM to Tuffier's line was 23.64mm when prone and 25.47 mm when flexed, constituting a statistically significant increase in the distance (P=0.0061). Therefore, in the absence of advanced imaging modalities, Tuffier's line provides practitioners with a simple method of determining a level caudal to the termination of the spinal cord, at approximately the L4/L5 in a prone neonate and the upper margins of L5 when flexed. Clin. Anat. 27:370–375, 2014. © 2013 Wiley Periodicals, Inc.