Objective: Reticular basement membrane (RBM) thickness is one of the pathological features of asthma and can be measured in endobronchial biopsies. We assessed the feasibility of endobronchial biopsies in a routine clinical setting and investigated the clinical value of RBM thickness measurements for asthma diagnosis in children. Methods: We included all children who underwent bronchoscopy with endobronchial mucosal biopsies for clinical reasons and divided them into three subgroups: (1) no asthma, (2) mild-moderate asthma, and (3) problematic severe asthma. Results: In 152/214 (71%) patients, mean age 9.5 years (SD 4.6; range 0.1-18.7) adequate biopsies were retrieved in which RBM thickness could be measured. Mean (SD) RBM thickness differed significantly among children without asthma, with mild-moderate asthma, and with problematic severe asthma (p = 0.04), 4.68 (1.24) μm, 4.56 (0.89) μm, and 5.21 (1.10) μm respectively. This difference disappeared after adding exhaled nitric oxide to the multivariate model. Conclusions: This study confirms the difference in RBM thickness between children with and without asthma and between asthma severities in a routine clinical care setting. However, quantifying the RBM thickness appeared to have no added clinical diagnostic value for asthma in children.