Type 2 diabetes (T2D) is a complex metabolic disorder with a strong genetic component but the understanding of genetic variants that predispose to T2D remain largely unknown. Here we studied diabetes susceptibility in a next-generation genetic reference population, generated by a specialized multi-parental scheme for studying complex traits and the crosstalk between genetic and environmental factors called the Collaborative Cross (CC). We aimed to identify and fully characterize a number of diabetes-susceptible CC mouse strains in order to uncover the novel pathogenesis of T2D using the CC. Random blood glucose was taken from fifty CC strains at 8-10 weeks of age as a phenotypic screening to determine hyperglycaemic CC strains. We identified two hyperglycaemic lines PIPING_BD (random blood glucose 23.0 ± 1.5 mM) and PUB_CD (random blood glucose 17.3 ± 0.8 mM) as diabetes susceptible strains. In this study, we performed intravenous glucose tolerance tests (IVGTT), oral glucose tolerance tests (OGTT) on 10-12 week old PIPING_BD, PUB_CD and C57BL6 control mice to evaluate glucose tolerance and in vivo insulin secretion. Histological analysis was conducted on resected pancreata taken at 10-12 weeks of age. In addition, ex vivo islet insulin secretion on PIPING_BD, PUB_CD and the C57BL/6 control mice in response to 2.8mM and 20mM glucose stimulation were conducted to assess b-cell function. Our results showed PIPING_BD and PUB_CD mice had impaired insulin secretion and glucose intolerance compared with C57BL/6 control. Interestingly, PIPING_BD and PUB_CD mice have normal islet number, size and pancreatic insulin staining. Moreover, ex vivo islet insulin secretion experiments showed that PIPING_BD and PUB_CD have normal insulin secretion compared with C57BL6 controls. Our data indicate that these hyperglycaemic CC lines displayed poor glucose tolerance and impaired insulin secretion in vivo but normal islet morphology, number and functional glucose-stimulated insulin secretion (GSIS) in vitro, which could provide novel mechanisms that underlie hyperglycaemia in T2D subjects and a new insight of T2D aetiology.