Background HIV-2 prevalence is poorly understood outside West Africa. HIV-2 infection is characterized by lower viremia, lower risk transmission and slower disease progression compared to HIV-1. Most diagnostic tests target both HIV1 and 2 but do not differentiate between the two and as such most infections in HIV-1 predominant areas are presumed to be HIV-1. Under detection of HIV-2 especially using diagnostic methods that are biased towards HIV-1 may pose a risk of hampering the epidemic control efforts. HIV-2 is responsible for an attenuated infection compared to HIV-1 thus, HIV-2 infection is characterized by low and frequently undetectable viral loads and a slower course to AIDS. In Uganda, we recently transitioned to the Roche Cobas 8800/6800 using the cobas HIV-1/HIV-2 Qualitative nucleic acid test, for the early infant diagnosis of HIV. The platform has the ability to differentiate between HIV-1 and HIV-2 to detect presence of HIV-2 in DBS, Serum, Plasma and whole blood samples. Method Twenty-four thousand six hundred and nineteen (24,619) Dry blood spots (DBS) collected from infants born to mothers living with HIV AIDs in Uganda were tested according to the Uganda consolidated guidelines for HIV prevention and treatment. All these infants were below 18 months of age. These were tested for routine early infant diagnosis (EID) at the centralized reference lab as per the Uganda national HIV consolidated guideline. Testing was performed using the cobas HIV-1/HIV-2 Qualitative nucleic acid test for use on the cobas 5800/6800/8800 Systems. This test is an in vitro nucleic acid amplification test for the qualitative detection and differentiation of human immunodeficiency virus (HIV) type 1 (HIV-1) and type 2 (HIV-2) in human serum, plasma, and dried blood spots (DBS). The specificity of cobas HIV-1/HIV-2 Qualitative is 100% (95% confidence limit: > 99.5%). This test is both FDA approved and WHO Prequalified. Results Of the 24,619 DBS tested between May and November 2023, 443 were confirmed positive (1.9%). Four (4) of the 466 (0.9%) were confirmed HIV-2. These 4 are from Kampala (1), Kagadi (1), Namutumba (1), and Oyam (1) districts. The HIV-2 positive samples had higher ct values (39.2, 40.33, 40.35, 44.62) very low viral load compared to the average ct value of less than 30 for the HIV-1 positive samples and are representative of the 95th percentile. Discussion We report for the first time, the likely mother to child transmission of HIV-2, with 4 cases reported between May to November 2023. The HIV-2 positive samples presented with high CT values, indicative of low viral load that is characteristic for HIV-2. Further investigations are ongoing to gather more details about the HIV-2 positive infants and to perform alternative tests to rule put non-specificity.