Type-2 diabetes (T2D) is a complex metabolic disease characterized by impaired glucose tolerance. Despite environmental high risk factors, host genetic background is a strong component of T2D development. Herein, novel highly genetically diverse strains of collaborative cross (CC) lines from mice were assessed to map quantitative trait loci (QTL) associated with variations of glucose-tolerance response. In total, 501 mice of 58 CC lines were maintained on high-fat (42 % fat) diet for 12 weeks. Thereafter, an intraperitoneal glucose tolerance test (IPGTT) was performed for 180 min. Subsequently, the values of Area under curve for the glucose at zero and 180 min (AUC0-180), were measured, and used for QTL mapping. Heritability and coefficient of variations in glucose tolerance (CVg) were calculated. One-way analysis of variation was significant (P < 0.001) for AUC0-180 between the CC lines as well between both sexes. Despite Significant variations for both sexes, QTL analysis was significant, only for females, reporting a significant female-sex-dependent QTL (~2.5 Mbp) associated with IPGTT AUC0-180 trait, located on Chromosome 8 (32-34.5 Mbp, containing 51 genes). Gene browse revealed QTL for body weight/size, genes involved in immune system, and two main protein-coding genes involved in the Glucose homeostasis, Mboat4 and Leprotl1. Heritability and coefficient of genetic variance (CVg) were 0.49 and 0.31 for females, while for males, these values 0.34 and 0.22, respectively. Our findings demonstrate the roles of genetic factors controlling glucose tolerance, which significantly differ between sexes requiring independent studies for females and males toward T2D prevention and therapy.