Introduction: While limited longitudinal studies have assessed the stability of the gut microbiota in fecal samples, there are no published studies to date that have examined the stability of the mucosa-associated microbiota. In this study, we aimed to concurrently characterize the variability in the gut microbiota in mucosal biopsies and stool samples of healthy subjects over a 9 month period. Methods: Healthy control subjects (n=9) were recruited from the community and underwent 4 unprepped flexible sigmoidoscopies at 0, 3, 6, and 9 months. Biopsies were taken of the colonic mucosa during the sigmoidoscopy and stool samples were collected at the same visit. Microbiota was characterized by sequencing of the 16S rDNA on a 454 Roche Titanium platform. Obtained sequences were filtered for quality/chimeras, and were analyzed using Qiime. Unifrac distances were calculated between the samples of the same subject (intra-individual distances), and were compared to the distances from the samples of other subjects (inter-individual distances). Results: Bacterial composition remained relatively stable over time in individual subjects over 9 months; and inter-individual Unifrac distances were higher than intra-individual ones ( 0.714 ± 0.058 vs.0.610 ± 0.085 respectively, p=0.001) for the entire sample set. This significant difference between inter-individual and intra-individual Unifrac distances was preserved for both colonic biopsy samples and for stool samples (p=0.0001 for both). When intra-individual Unifrac distances between stool samples were compared to that of biopsy samples, there was no observed difference between the sample types (0.586 ± 0.628 vs. 0.579 ± 0.076 respectively, p=0.384), suggesting that there is a similar degree of variability in the longitudinal stool samples and the longitudinal biopsy samples of the same individual. However, higher inter-individual Unifrac distances were noted in longitudinal stool samples compared to longitudinal biopsy samples (0.713 ± 0.057 vs. 0.691 ± 0.052 respectively, p=0.0001), suggesting that there is a slightly lower variability within longitudinal biopsy samples of different individuals. Conclusion: Inter-individual variation in the bacterial microbiome is preserved in longitudinal colonic biopsy samples. Time and sampling related variability in the mucosa-associated colonic bacterial microbiome in healthy individuals is not large enough to supercede inter-individual differences.