Obesity has emerged as a major global health problem and is associated with various diseases, such as metabolic syndrome, type 2 diabetes mellitus, and cardiovascular diseases. The inbred C57BL/6 mouse strain is often used for various experimental investigations, such as metabolic research. However, over time, genetically distinguishable C57BL/6 substrains have evolved. The manifestation of genetic alterations has resulted in behavioral and metabolic differences. In this study, a comparison of diet-induced obesity in C57BL/6JHanZtm, C57BL/6NCrl and C57BL/6 J mice revealed several metabolic and immunological differences such as blood glucose level and cytokine expression, respectively, among these C57BL/6 substrains. For example, C57BL/6NCrl mice developed the most pronounced adiposity, whereas C57BL/6 J mice showed the highest impairment in glucose tolerance. Moreover, our results indicated that the immunological phenotype depends on the intestinal microbiota, as the cell subset composition of the colon was similar in obese ex-GF B6NRjB6JHanZtm and obese B6JHanZtm mice. Phenotypic differences between C57BL/6 substrains are caused by a complex combination of genetic and microbial alterations. Therefore, in performing metabolic research, considering substrain-specific characteristics, which can influence the course of study, is important. Moreover, for unbiased comparison of data, the entire strain name should be shared with the scientific community.