The effect of genetic susceptibility to the pathogenesis of type 2 diabetes is significant, whereas the identity of the common genetic variants with a large effect on glucose dysregulation have not yet been fully defined. Our study aims to identify the genetic variants influencing glucose homeostasis in a diverse genetic mouse population called the Gene Mine. A phenotypic screening for glycaemic variability was conducted across 53 inbred strains at 8 weeks of age. Random blood glucose levels were measured and subjected to automated genome wide mapping. Two loci with NZO derived variants on chromosome 7 were significantly associated with hyperglycaemia (P=3.682e-15), both of which have been associated with impairment of insulin secretion to some extent in human studies. To further narrow down the positional candidate and explore the physiological role of these genetic variants in glucose homeostasis, we performed in depth characterisation of two hyperglycaemic strains which have the deleterious alleles and determined gene expression of all potential candidate genes in the susceptible versus non-susceptible strains. Results showed that these hyperglycaemic strains presented a diabetic phenotype with overt glucose intolerance following an oral glucose tolerance test and significant impairment in glucose stimulated insulin secretion compared to control C57BL/6 mice. Interestingly, this marked reduction in insulin secretion was associated with no differences in islet size, area and morphology, indicating that these variants lead to alterations in beta cell functions. Using gene expression analysis, we identified two novel genes in the two loci and preliminary gene-knockdown experiments in MIN6 cells suggest that they directly impact insulin secretion. Our results highlight the advantages of using the Gene Mine mice to rapidly discover and elucidate the function of novel genes predisposing to hyperglycaemia and impaired insulin secretion. These experiments may provide valuable information to identifying gene targets for the management of type 2 diabetes.