Insulin dynamically modulates protein phosphorylation to promote glucose uptake into metabolic tissues. The failure of this process – insulin resistance – is a harbinger of diseases including type 2 diabetes. In humans, skeletal muscle is the most important site of insulin resistance. An individual’s genetic background and diet are two of the strongest determinants of insulin sensitivity at this site. However, we lack a systematic understanding of how genetic variation and dietary quality modulate insulin phosphorylation networks in muscle, and how this culminates in insulin resistance. To address this knowledge gap we developed a method to interrogate mouse skeletal muscle in vivo, which leverages data-independent-acquisition-based phosphoproteomics to provide global snapshots of insulin signalling and measures insulin-stimulated glucose uptake from a single tissue. We applied this method to five genetically distinct inbred mouse strains fed a standard chow diet or a high-fat high-sucrose diet (HFD), and observed pronounced effects of genetic background and diet on muscle glucose uptake. Phosphoproteomics revealed marked rewiring of insulin signalling by genetics and diet. Of the 441 phosphopeptides that responded to insulin, half responded to insulin with different strength between strains and a quarter were affected by HFD-feeding. Genetic background modulated the magnitude and direction of nearly all HFD effects. Finally, association of signalling changes with glucose uptake prioritised phosphosites potentially involved in insulin resistance, including activating sites on the glycolytic enzyme Pfkfb2 and the nitric oxide synthase Nos3. These results demonstrate the fundamental role of genetics in shaping signalling networks, their responses to perturbation, and their phenotypic outputs.