Objectives: The beta-D-glucan assay (BDG) has been added to the EORTC/MSG criteria for the diagnosis of invasive fungal infections (IFI), but data from pediatric populations is scarce. The aim of this study was to evaluate performance of BDG in a cohort of hemato-oncological children with hematological malignancy at risk for IFI. Methods: 113 patients were included through an 18-month period. In addition to routine IFI screening, BDG was assayed once a week. IFIs were classified using EORTC/MSG criteria without including the BDG results. Performances were assessed after a ROC analysis for optimization and multivariate analysis to detect the causes of false positivity. Results: 8 proven and 4 probable IFIs, and 7 possible IFIs were diagnosed in 9 and 7 patients, respectively. Sensitivity and specificity increased from 75% and 56% to 100% and 91.1%, respectively when considering the whole population and patients not having received any antifungals prior to the test. Multivariate analysis revealed that being younger than 7, severe colitis/mucositis, recent administration of polyvalent immunoglobulins and digestive colonization with Enterococcus sp were independent risk factors for false positivity. Conclusions: BDG is a valuable test to detect IFI in pediatric patients not previously treated with antifungals and to detect the occurrence of chronic infection. (C) 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.