The C57BL/6 mouse is an established inbred mouse stain, utilised for studies into obesity and metabolic dysfunction. While it is known that different mouse strains can have distinct metabolic phenotypes, it is possible that individual C57BL/6 substrains may also differ in their metabolism. We investigated the metabolic phenotype of two C57BL/6J colonies originally obtained from the Jackson’s Laboratory but maintained separately and for different lengths of time at the same  external animal facility (the Alfred Medical Research & Education Precinct Animal Services, Australia (AMREP-AS)). Interestingly, our phenotyping uncovered significant differences between the substrains designated “C57BL/6JA” for the colony that had been bred for longer at AMREP-AS and “C57BL/6J” for the colony that had been established more recently. On a normal chow diet the C57BL/6JA mice had greater lean body mass, as well as improved glucose and insulin tolerance that was surprisingly accompanied with hyperinsulinemia. The C57BL/6JA mice also tended to have increased liver size which was associated with increased hepatic ceramide accumulation, particularly the longer chain ceramide species. To identify whether these alterations were due to genetic drift between the substrains, we analysed liver RNA samples from each group using microarray analysis. This analysis identified one single gene that was differentially expressed between the two substrains acad10, a gene that encodes a member of the acyl-CoA dehydrogenase family of enzymes (ACADs), which participate in the beta-oxidation of fatty acids. This data demonstrates the inherent metabolic differences that can develop over a short period of time even in mice maintained in the same environment and originally sourced from the same supplier. This study suggests caution should be used in using the correct C57BL/6 substrain controls and in the interpretation of data from laboratories using different C57BL/6 substrains.