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Background: We sought to determine if vertebral interspace selection for performance of infant lumbar puncture (LP) varies between less experienced trainees and more experienced pediatric emergency medicine (PEM) attending physicians., Methods: We performed an observational prospective study using a convenience sample of infants aged 0 to 12 months presenting to a single emergency department. Trainees with limited LP experience (defined as less than 10 infant LPs performed) marked their preferred LP insertion site with an invisible ultraviolet pen. PEM attending physicians subsequently marked their preferred LP insertion site with a visible pen. A trained sonographer then performed a bedside ultrasound to confirm interspace concordance or discordance. Our primary outcome was the proportion of concordant marked insertion sites., Results: Of the 110 patients enrolled, 102 (92.8%) completed study procedures. Trainee and PEM attending LP interspace markings were concordant in 27% of cases. Trainees marked a preferred interspace below the level of the attending in 55% of patients: 29 (28.4%) marked one spot inferior, 20 (19.6%) marked two spots inferior, and seven (6.9%) marked three spots inferior in relation to the attending., Conclusions: There is variability of preferred LP insertion site based on provider experience. Trainees with limited LP experience tended to mark insertion spaces more caudal than those marked by the attending physicians in an area where the subarachnoid space is slightly smaller., (© 2019 by the Society for Academic Emergency Medicine.)