The reliability of lipidomics, an approach to identify the presence and interactions of lipids, to analyze the bone marrow lipid composition among pediatric populations with bone fragility is unknown. The objective of this study was to assess the test-retest reliability, standard error of measurement (SEM), and the minimal detectable change (MDC) of vertebral bone marrow lipid composition determined by targeted lipidomics among children with varying degrees of bone fragility undergoing routine orthopedic surgery. Children aged 10 to 19 years, with a confirmed diagnosis of adolescent idiopathic scoliosis (n = 13) or neuromuscular scoliosis and cerebral palsy (n = 3), undergoing posterior spinal fusion surgery at our institution were included in this study. Transpedicular vertebral body bone marrow samples were taken from thoracic vertebrae (T11, 12) or lumbar vertebrae (L1 to L4). Lipid composition was assessed via targeted lipidomics and all samples were analyzed in the same batch. Lipid composition measures were examined as the saturated, monounsaturated, and polyunsaturated index and as individual fatty acids. Relative and absolute test-retest reliability was assessed using the intraclass correlation coefficient (ICC), SEM, and MDC. Associations between demographics and index measures were explored. The ICC, SEM, and MDC were 0.81 (95% CI, 0.55-0.93), 1.6%, and 4.3%, respectively, for the saturated index, 0.66 (95% CI, 0.25-0.87), 3.5%, and 9.7%, respectively, for the monounsaturated index, and 0.60 (95% CI, 0.17-0.84), 3.6%, and 9.9%, respectively, for the polyunsaturated index. For the individual fatty acids, the ICC showed a considerable range from 0.04 (22:2n-6) to 0.97 (18:3n-3). Age was positively correlated with the saturated index (r ² = 0.36; p = 0.014) and negatively correlated with the polyunsaturated index (r ² = 0.26; p = 0.043); there was no difference in index measures by sex (p > 0.58). The test-retest reliability was moderate-to-good for index measures and poor to excellent for individual fatty acids; this information can be used to power research studies and identify measures for clinical or research monitoring. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.