Aims/hypothesis: Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia. Methods: Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference. Results: Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the E2f8 (p = 2.19 × 10−15) and Dlg2 loci (p = 3.83 × 10−8) on chromosome 7 to be significantly associated with hyperglycaemia susceptibility. Fine mapping the implicated regions using congenic mice demonstrated that these two loci have independent effects on insulin secretion in vivo. Significantly, our results revealed that increased E2F8 and DLG2 gene expression are correlated with enhanced insulin secretory function in human islets. Furthermore, loss-of-function studies in MIN6 cells demonstrated that E2f8 is involved in insulin secretion through an ATP-sensitive K+ channel-dependent pathway, which leads to a 30% reduction in Abcc8 expression. Similarly, knockdown of Dlg2 gene expression resulted in impaired insulin secretion in response to glucose and non-glucose stimuli. Conclusions/interpretation: Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of E2f8 and Dlg2 as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases.