Introduction: Adult women with Turner syndrome (TS) have a high prevalence of diabetes and β-cell dysfunction that increases morbidity and mortality, but it is unknown if there is β-cell dysfunction present in youth with TS. This study aimed to determine the prevalence of β-cell dysfunction in youth with TS and the impact of traditional therapies on insulin sensitivity (SI) and insulin secretion. Methods: Cross-sectional, observational study recruited 60 girls with TS and 60 healthy controls (HC) matched on pubertal status. Each subject had a history, physical exam, and oral glucose tolerance test (OGTT). Oral glucose and c-peptide minimal modeling was used to determine β-cell function. Results: Twenty-one TS girls (35%) met criteria for prediabetes. Impaired fasting glucose was present in 18% of girls with TS and 3% HC ( p value = 0.02). Impaired glucose tolerance was present in 23% of TS girls and 0% HC ( p value <0.001). The hemoglobin A1c was not different between TS and HC (median 5%, p = 0.42). Youth with TS had significant reductions in SI, β-cell responsivity (Φ), and disposition index (DI) compared to HC. These differences remained significant when controlling for body mass index z-score ( p values: 0.0006, 0.002, <0.0001 for SI, Φ total, DI, respectively). Conclusions: β-Cell dysfunction is present in youth with TS compared to controls. The presence of both reduced insulin secretion and SI suggest a unique TS-related glycemic phenotype. Based on the data from this study, we strongly suggest that providers employ serial OGTT to screen for glucose abnormalities in TS youth.