Genetics and diet play a major role in the development of insulin resistance (IR). While diet effects have been extensively studied, the role of genetics alone or the interaction between genes and the environment is not well characterized. This is due, in part, to the inability to subject the same background to multiple environmental perturbations, and the lack of tissue-specific phenotypic resolution afforded by many model systems. The latter is crucial, as it is widely accepted that, under obesogenic conditions, IR in muscle is driven by IR in adipose tissue via some form of tissue crosstalk. However, there is a need to investigate how genetic background influences the development of tissue-specific IR and consequent systemic disease. We undertook a comprehensive analysis of tissue level insulin action combined with other metabolic phenotypes in a panel of inbred mice strains fed two different diets. Each strain exhibited unique metabolic responses to Western Diet (WD). Strikingly, muscle and adipose tissue IR were not correlated, with IR in each tissue being associated with discrete metabolic indices: muscle IR, but not AT IR, was strongly correlated with hyperinsulinemia; soleus IR was correlated with fat pad mass, indicating a possible link between lipid storage capacity and muscle IR independently of AT IR per se. In contrast to soleus IR, adipose IR was correlated with adipocyte hypertrophy but not fat pad mass, suggesting AT IR is driven by factors intrinsic to the adipocyte. Interestingly, the heritability of IR was higher in EDL than soleus, whereas Western diet feeding had a greater impact on IR in soleus, emphasizing mechanistic heterogeneity behind IR in different muscles, which may be based on functional and structural differences. Co-expression network analysis of the soleus proteome revealed marked variation in the expression levels of specific proteins across different inbred strains and in response to diet. The strongest response module comprised numerous glycolytic enzymes that were strongly correlated with muscle IR. By leveraging the effects of discrete dietary conditions across diverse genetic backgrounds in mice, this study has uncovered extensive gene-by-diet interactions, which create unique tissue- and strain-specific IR phenotypes. We have harnessed these signatures to deconvolute the metabolic contributions of tissue-specific IR, revealing unique AT-muscle crosstalk, as well as potential mechanisms of IR inherent to each tissue.