C57BL/6J-related mouse strains are widely used animal models for diet-induced obesity (DIO). Multiple vendors breed C57BL/6J-related substrains which may introduce genetic drift and environmental confounders such as microbiome differences. To address potential vendor/substrain specific effects, we compared DIO of C57BL/6J-related substrains from three different vendors: C57BL/6J (Charles Rivers), C57BL/6JBomTac (Taconic Bioscience) and C57BL/6JRj (Janvier). After local acclimatization, DIO was induced by either a high-fat diet (HFD, 60% energy from fat) or western diet (WD, 42% energy from fat supplemented with fructose in the drinking water). All three groups on HFD gained a similar amount of total body weight, yet the relative amount of fat percentage and mass of inguinal- and epididymal white adipose tissue (iWAT and eWAT) was lower in C57BL/6JBomTac compared to the two other C57BL/6J-releated substrains. In contrast to HFD, the three groups on WD responded differently in terms of body weight gain, where C57BL/6J was particularly prone to WD. This was associated with a relative higher amount of eWAT, iWAT, and liver triglycerides. Although the HFD and WD had significant impact on the microbiota, we did not observe any major differences between the three groups of mice. Together, these data demonstrate significant differences in HFD- and WD-induced adiposity in C57BL/6J-related substrains, which should be considered in the design of animal DIO studies.