The role of aspiration-associated extraesophageal reflux disease (AERD) in patients with chronic respiratory symptoms is not well defined. Identifying the frequency of AERD in these patients may provide guidance in their treatment. To determine the prevalence of AERD in patients with chronic respiratory symptoms and to assess the utility of pepsin as a new marker for AERD. Case-control study performed from 2008 through 2012.Western blot analysis for pepsin and oil red O staining for lipid-laden macrophages (LLMs) was performed on bronchoalveolar lavage fluid specimens. Tertiary referral center. Sixty-five patients (aged 4.5 months to 24 years) with chronic pulmonary disease, with or without tracheostomy, were compared with controls undergoing elective surgery who had no history of pulmonary disease. Presence of pepsin and LLMs and quantity of LLMs in specimens. Seventy-six total patients participated: 34 patients who underwent bronchoscopy, 31 patients with tracheostomy, and 11 controls. Pepsin-positive bronchoalveolar lavage fluid specimens were identified in 25 patients who underwent bronchoscopy (74%) and 22 patients with tracheostomy (71%). All specimens from controls were negative for pepsin. Presence of LLMs was identified in specimens from 31 patients in the bronchoscopy group (91%), 16 patients in the tracheostomy group (52%), and 7 controls (64%), with a similar distribution of the quantity of LLMs in each lavage fluid specimen among the groups. Patients with chronic pulmonary disease have a high prevalence of AERD, which may have important treatment implications. The presence of pepsin was a better predictor of AERD in patients with respiratory symptoms compared with controls than presence of LLMs. Detection of pepsin in bronchoalveolar lavage fluid specimens can serve as a biomarker for AERD and is potentially superior to the current method of measuring LLMs. Whereas there is a significant association between AERD and the presence of chronic respiratory symptoms, this study does not verify causation. Additional study investigating the mechanism of pepsin on the respiratory epithelium may further our understanding of the pathophysiologic characteristics of this association and provide additional management options for these patients.