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Summary: The intestinal microbiota is a fundamental factor that broadly influences physiology. Thus, studies using transgenic animals should be designed to limit the confounding effects of microbiota variation between strains. Here, we report the impact on intestinal microbiota of co-housed versus F2-generation littermates, two commonly used techniques to standardize microbiota in animal models. Our results establish that while fecal microbiota is partially normalized by extended co-housing, mucosal communities associated with the proximal colon and terminal ileum remain stable and distinct. In contrast, strain inter-crossing to generate F2 littermates allows robust microbiota standardization in fecal, colon, and ileum sampling locations. Using reciprocal inter-crosses of P1 parents, we identify dissymmetry in F2 community structures caused by maternal transmission, in particular of the Verrucomicrobiaceae. Thus, F2 littermate animals from a unidirectional P1 cross should be used as a standard method to minimize the influence of the microbiota in genotype-phenotype studies. : Standardization of the microbiota in mouse models ensures reproducibility of findings and avoids erroneous conclusions. Here, Robertson et al. demonstrate that the use of F2-generation littermates is the superior method for standardization of the gut microbiota between experimental groups to minimize the influence of the microbiota in genotype-phenotype studies. Keywords: microbiota, microbiota standardization, intestinal, co-housing, littermates, mouse