Rationale Mucus hyper-secretion is a hallmark feature of asthma and other obstructive airway diseases. Two mucins, MUC5AC and MUC5B, represent the major glycoprotein components of mucus. Little is known about the genetic determinants of MUC5AC and MUC5B protein expression. We aim to identify genetic regulators of MUC5AC and MUC5B protein concentrations in a mouse model of asthma. Methods We applied a house dust mite allergen model of asthma to both founder lines (n=8) and incipient lines (n=154) of the Collaborative Cross (CC). We collected whole lung lavage samples 72 hours after allergen challenge and quantified MUC5AC and MUC5B by agarose gel electrophoresis followed by western blotting. Each CC mouse was genotyped on a high density Affymetrix array. Protein expression in lungs was examined using immunohistochemistry. Gene knockdown in vitro was accomplished using lentiviral constructs expressing shRNAs. Results CC founder lines sensitized and challenged with allergen exhibited statistically significant differences in both MUC5AC and MUC5B, providing evidence of heritability. Incipient CC lines exhibited a broad range of MUC5AC and MUC5B secretion, consistent with a polygenic architecture for these two phenotypes. Quantitative trait loci (QTL) for MUC5AC and MUC5B were identified on Chromosomes 13 (at 75 Mb) and 2 (at 154 Mb), respectively. We focused on the MUC5B QTL because of the large effect size and found that musculus-derived alleles were associated with lower MUC5B compared to alleles from domesticus and castaneus. We validated the difference in MUC5B concentrations due to QTL region genotype by performing a second set of experiments using independent CC lines of contrasting haplotypes (musculus vs. domesticus). Using additional gene expression and SNP datasets, we identified Bpifb1 as a candidate gene. We show that BPIFB1 expression colocalizes with MUC5B in airway epithelia and is upregulated by allergen treatment. Finally, we show that knockdown of BPIFB1 affects MUC5B secretion in primary human airway epithelial cells. Conclusions Concentrations of MUC5AC and MUC5B in the allergen-challenged lung are controlled by distinct genetic loci, and these loci are distinct from those that regulate mucin gene expression. Our data indicate that variation in Bpifb1 is strongly associated with MUC5B concentration after allergen challenge.