Abstract Background Bronchoalveolar lavage (BAL) has proven to be very useful to monitor the lung allograft after transplantation. In addition to allowing detection of infections, multiple BAL analytes have been proposed as potential biomarkers of lung allograft rejection or dysfunction. However, BAL collection is not well standardized and differences in BAL collection represent an important source of variation. We hypothesized that there are systematic differences between sequential BALs that are relevant to BAL analysis. Methods As part of 126 consecutive bronchoscopies in lung transplant recipients, two sequential BALs (BAL1 and BAL2) were performed in one location during each bronchoscopy by instilling and suctioning 50 ml of normal saline twice into separate containers. Cell concentration, viability and differentials, Surfactant Protein-D (SP-D), Club Cell Secretory Protein (CCSP), and levels of CXCL10, IL-10, CCL2, CCL5, VEGF-C, RAGE, CXCL9, CXCL1, IL-17A, IL-21, PDGF, and GCSF were compared between BAL1 and BAL2. Results Total cell concentration did not differ between BAL1 and BAL2; however, compared to BAL2, BAL1 had more dead cells, epithelial cells, neutrophils, and higher concentrations of airway epithelium-derived CCSP and inflammatory markers. BAL2 had a higher concentration of SP-D compared to BAL1. Conclusion In this study performed in lung transplant recipients, we show that sequential BALs represent different lung compartments and have distinct compositions. BAL1 represents the airway compartment with more epithelial cells, neutrophils, and epithelium-derived CCSP. Conversely, BAL2 samples preferentially the distal bronchoalveolar space with greater cell viability and higher SP-D. Our findings illustrate how the method of BAL collection can influence analyte concentrations and further emphasize the need for a standardized approach in translational research involving BAL samples.