In a recent study examining the response of different mouse strains to high-fat feeding, we showed that C57BL/6, 129X1/SvJ, DBA/2 and FVB/N mice all became glucose intolerant and insulin resistant in response to a high-fat diet, while BALB/c mice were protected from the deterioration in glucose homeostasis and insulin action. Interestingly, all five mouse strains, including BALB/c mice, showed similar changes in many parameters that are commonly associated with glucose intolerance and insulin resistance, such as lipid accumulation in muscle, adipose tissue inflammation and markers of mitochondrial oxidative metabolism in various tissues. What differed in HFD-fed BALB/c mice was a lack of hepatic lipid accumulation. To explore the full extent of differences in lipid composition in these 5 mouse strains, we have performed lipidomics analysis of liver and skeletal muscle. In muscle, all 5 strains exhibited similar changes in triacylglycerol (TAG), diacylglycerols (DAG), ceramide (Cer) and sphingomyelin (SM) species after high-fat feeding. In contrast, in the liver BALB/c mice were the only strain that did not accumulate any excess TAG and DAG. In addition, the most exciting finding was that the favourable metabolic profile of BALB/c mice was associated with a preferential accumulation of very long-chain Cer and SM species (C>22) over long-chain species (mainly 16:0 and 18:0), while the opposite pattern was observed in the mouse strains that developed diet-induced glucose intolerance and insulin resistance. These sphingolipid differences are likely related to changes in the expression of specific ceramide synthase (CerS) isoforms and the fatty acid elongase Elovl1 in the liver and suggest a potentially important therapeutic role for targeted manipulation of specific sphingolipid species in the treatment of insulin resistance.