The gut microbiota (GM) may influence disease expression in several animal models for inflammatory diseases. It may therefore seem reasonable to pursue reduction in the number of animals used for individual studies by reducing the variation in the GM. Previous studies have shown that the composition of the GM is related to genetics to a certain extent. We hypothesized that the GM similarity in a group of mice born by mothers not being sisters would be lower than that in a group born by mothers being sisters. The lower similarity could lead to clustering of the GM of mice born by non-sisters according to their mothers, while such clustering would not be visible if the mothers were sisters. We used 16S rRNA gene (V3 region) polymerase chain reaction-derived amplicon profiling by denaturing gradient gel electrophoresis (DGGE) to study the GM composition in caecum samples of 33 eight-week-old C57BL/6Sca mice from a breeding set-up with dam breeders that were sisters, as well as caecum samples of 35 eight-week-old C57BL/6Sca mice from a breeding set-up with dam breeders that were not sisters. Principal component analysis revealed a significant difference between the litters from the breeding set-up with dam breeders that were not sisters, whereas no significant difference between the litters based on the breeding set-up with dam breeders that were sisters was observed. The results obtained indicate that the systematic variation in the GM of inbred mice can be reduced by increasing the family relatedness of the breeding pairs.