Background: Emerging evidence suggests a possible association between hyperglycemia and dolutegravir (DTG), a preferred first-line antiretroviral agent in sub-Saharan Africa. There is need for rigorous studies to validate this association in the face of increasing DTG use and burden of non-communicable diseases among people living with HIV (PLHIV). Methods: We conducted a case-control study in Kampala, Uganda to assess the risk of hyperglycemia associated with DTG-based antiretroviral therapy. Cases were PLHIV with hyperglycemia and controls were PLHIV without hyperglycemia confirmed by fasting plasma glucose and 2-hour oral glucose tolerance tests. Data were collected using interviewer administered questionnaires and medical record abstraction. Analysis compared cases and controls on DTG use prior to diagnosis of hyperglycemia while controlling for potential confounders using multivariable logistic regression. Findings: A total 435 participants were included (204 were cases, 231 controls). In multivariable analysis, patients with prior DTG use had nearly 30-fold greater odds of hyperglycemia compared to those who had non-DTG based regimens (adjusted odds ratio [aOR] 29·06, 95% CI 9·86–85·70). The odds of hyperglycemia also increased with age (56 vs. 18–35 years,aOR 7·53, 95% CI 2·65–21·38), and hypertension (aOR 5·64, 95% CI 2·60–12·21). Interpretation: Our study demonstrates a strong association between prior DTG exposure and hyperglycemia. Given the benefits of DTG, the wide-scale roll-out in sub-Saharan Africa, and the growing burden of diabetes mellitus (DM), there is need for systematic screening for hyperglycemia prior to initiating DTG, routine glucose monitoring, and the consideration of alternate regimens for those at risk for DM. Funding: This research was supported by the US National Institutes of Health (NIH), Fogarty International Center and Global Health Equity Scholars(D43TW010540). Declaration of Interests: The authors declare they have no conflict of interest. Ethics Approval Statement: The study protocol was reviewed and approved by the Institutional Review Boards of The AIDS Support Organization (TASO), Uganda, he Uganda National Council of Science and Technology (UNCST) and Yale University. All procedures performed in studies involving human participants were following the ethical standards of the institutional and/or national research committee. We obtained written Informed consent from all participants.