High mobility group protein B1 (HMGB1) has been implicated as an important mediator in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). However, the expression of HMGB1 in plasma and sputum of patients with asthma and COPD across disease severity needs to be defined. The objective of the study was to examine the induced sputum and plasma concentrations of HMGB1 in COPD and asthmatic patients to determine differences in HMGB1 levels between these diseases and their relationship with airway obstruction and inflammatory patterns. A total of 147 participants were enrolled in this study. The participants included 34 control subjects, 61 patients with persistent asthma (according to the Global Initiative for Asthma [GINA] guidelines) and 47 patients with stable COPD (stratified by Global Initiative for Chronic Obstructive Lung Disease [GOLD] status). Spirometry was performed before sputum induction. HMGB1 levels in induced sputum and plasma were determined by enzyme-linked immunosorbent assay. Sputum and plasma concentrations of HMGB1 in patients with asthma and COPD were significantly higher than concentrations in control subjects and were significantly negatively correlated with forced expiratory volume in 1 s (FEV(1)), FEV(1) (% predicted) in all 147 participants. The levels of HMGB1 in induced sputum of COPD patients were significantly higher than those of asthma patients and healthy controls (P < 0.001). This difference was present even after adjusting for sex, age, smoking status, daily dose of inhaled corticosteroids and disease severity. There were no significant differences in HMGB1 levels between patients with eosinophilic and noneosinophilic asthma. HMGB1 levels in asthmatic and COPD patients were positively correlated with neutrophil counts and percentage of neutrophils. In multivariate analysis, the two diseases (asthma and COPD) and disease severity were independent predictors of sputum HMGB1, but not smoking, age or use of inhaled corticosteroids. In conclusion, these data support a potential role for HMGB1 as a biomarker and diagnostic tool for the differential diagnosis of asthma and COPD. The importance of this observation on asthma and COPD mechanisms and outcomes should be further investigated in large prospective studies.