Chronic rhinosinusitis (CRS) is a common and potentially debilitating disease characterized by inflammation of the sinus mucosa for longer than 12 weeks. Bacterial colonization of the sinuses and its role in the pathogenesis of this disease is an ongoing area of research. Recent advances in culture-independent molecular techniques for bacterial identification have the potential to provide a more accurate and complete assessment of the sinus microbiome, however there is little concordance in results between studies, possibly due to differences in the sampling location and techniques. This study aimed to determine whether the microbial communities from one sinus could be considered representative of all sinuses, and examine differences between two commonly used methods for sample collection, swabs and tissue biopsies. High-throughput DNA sequencing of the bacterial 16S rRNA gene was applied to both swab and tissue samples from multiple sinuses of 19 patients undergoing surgery for treatment of CRS. Results from swabs and tissue biopsies showed a high degree of similarity, indicating that swabbing is sufficient to recover the microbial community from the sinuses. Microbial communities from different sinuses within individual patients differed to varying degrees, demonstrating that it is possible for distinct microbiomes to exist simultaneously in different sinuses of the same patient. The sequencing results correlated well with culture-based pathogen identification conducted in parallel, although the culturing missed many species detected by sequencing. This finding has implications for future research into the sinus microbiome, which should take this heterogeneity into account by sampling patients from more than one sinus. It may also be of clinical importance, as determination of antibiotic sensitivities using culture of a swab from a single sinus could miss relevant pathogens that are localized to another sinus.