Childhood obesity is a global public health threat, with an alarming rise in incidence. Obesity at young age has short-term and long-term morbidity. It is, therefore, important to accurately assess body composition throughout infancy and childhood to identify excess adiposity. However, reference values for age 2-5 years, needed to interpret measurements and identify young children at risk, are lacking. Our primary objective was to fill the current gap in reference values by constructing sex-specific body composition reference values and charts for fat mass (FM), fat mass percentage (FM%), fat mass index (FMI), lean body mass (LBM), lean body mass index (LBMI) and total body less head bone mineral density (BMDTBLH) for children aged 2-5 years using Dual-Energy X-ray Absorptiometry (DXA). We performed 599 accurate DXA-measurements in 340 term-born children aged 2-5 years, using Lunar Prodigy with Encore software (V14.1). Using GAMLSS, sex-specific reference values and charts were created for FM, FM%, FMI, LBM, LBMI and BMDTBLH. Sex-specific body composition reference values and charts for age 2-5 years were constructed. In boys and girls, FM and LBM increased from age 2-5 years (all p≤0.001), but body size-corrected FM% and FMI decreased (all p≤0.023). LBMI remained similar between 2 and 5 years of age. Girls had higher FM, FM% and FMI and lower LBM and LBMI compared to boys. BMC and BMDTBLH increased with age between 2 and 5 years of age (all p<0.001) and were similar for boys and girls. We present sex-specific reference values and charts for body composition and total body bone mineral density measured by DXA, based on a large cohort of healthy children aged 2-5 years. These longitudinal references can be used for clinical practice and research purposes to monitor body composition and bone mineral density development and identify children at risk for excess adiposity.