Transgenic and knockout mice are major research models used to investigate the aetiology of obesity and diabetes. However, genetically manipulated mice are produced on a range of genetic backgrounds and there is only limited information on the inherent propensity of different mouse strains to develop metabolic disease. To investigate strain-dependent differences in the susceptibility to diet-induced obesity and insulin resistance, we have carried out an extensive comparison of the response to a high-fat diet (HFD) of five commonly used mouse strains (DBA/2, 129X1/SvJ, BALB/c, C57BL/6 and FVB/N). Body weight was higher in four mouse strains (except BALB/c) in response to a HFD, while whole-body fat mass was significantly increased in all strains. Glucose tolerance was significantly impaired by the HFD in all strains except BALB/c, where the HFD animals displayed similar glucose tolerance to low-fat diet (LFD) counterparts. Consistent with these findings, insulin sensitivity was maintained at the level of LFD controls in fat-fed BALB/c mice, but was significantly reduced in all other strains. Tissue triglyceride (TAG) levels were assessed to determine if lipid accumulation in non-adipose tissue may be associated with the disparate effects of the HFD on insulin action. The DBA/2, 129X1/SvJ, C57BL/6 and FVB/N mice all displayed significant increases in skeletal muscle and liver TAG in response to the HFD. Intriguingly, BALB/c mice did not accumulate excess liver triglycerides, despite a similar increase in lipid deposition in muscle compared to other strains. Despite low liver triglycerides, BALB/c mice were the only strain that displayed increased liver lipoxidative damage, suggesting that triglyceride accumulation might prevent the formation of other lipid species that cause oxidative damage.This mouse strain comparison highlights the importance of genetic background in determining the metabolic response to HFD and shows that glucose tolerance correlates with lipid accumulation in the liver, but not in muscle.