Cystic fibrosis (CF) is a common fatal genetic disorder with mortality most often resulting from microbial infections of the lungs. Culture-independent studies of CF-associated microbial communities have indicated that microbial diversity in the CF airways is much higher than suggested by culturing alone. However, these studies have relied on indirect methods to sample the CF lung such as expectorated sputum and bronchoalveolar lavage (BAL). Here, we characterize the diversity of microbial communities in tissue sections from anatomically distinct regions of the CF lung using barcoded 16S amplicon pyrosequencing. Microbial communities differed significantly between different areas of the lungs, and few taxa were common to microbial communities in all anatomical regions surveyed. Our results indicate that CF lung infections are not only polymicrobial, but also spatially heterogeneous suggesting that treatment regimes tailored to dominant populations in sputum or BAL samples may be ineffective against infections in some areas of the lung.