Abstract Background: Colorectal cancer screening programs with fecal sample collection may provide a platform for population-based gut microbiome disease research. We investigated sample collection and storage method impact on the accuracy and stability of the V3-V4 region of the 16S rRNA genes and bacterial quantity across seven different collection methods [i.e., no solution, two specimen collection cards, and four types of fecal immunochemical test (FIT) used in four countries] among 19 healthy volunteers. Methods: Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the most abundant genera, alpha diversity metrics, and the first principal coordinates of the beta diversity matrices to estimate the stability of microbial profiles after storage for 7 days at room temperature, 4°C or 30°C, and after screening for the presence of occult blood in the stool. In addition, accuracy was estimated for samples frozen immediately compared to samples with no solution (i.e., the putative gold standard). Results: When compared with the putative gold standard, we observed significant variation for all collection methods. However, interindividual variability was much higher than the variability introduced by the collection method. Stability ICCs were high (≥0.75) for FIT tubes that underwent colorectal cancer screening procedures. The relative abundance of Actinobacteria (0.65) was an exception and was lower for different FIT tubes stored at 30°C (range, 0.41–0.90) and room temperature (range, 0.06–0.94). Conclusions: Paper-based collection cards and different types of FIT are acceptable tools for microbiome measurements. Impact: Our findings inform on the utility of commonly used fecal sample collection methods for developing microbiome-focused cohorts nested within screening programs.