The 2011 prevalence of human immunodeficiency virus (HIV) among pregnant women in Botswana was 30.4%. High coverage rates of HIV testing and antiretroviral prophylaxis have reduced the rate of mother-to-child transmission of HIV in Botswana from as high as 40% with no prophylaxis to <4% in 2011. In June 2005, the national Early Infant Diagnosis (EID) Program began testing HIV-exposed infants (i.e., those born to HIV-infected mothers) for HIV using polymerase chain reaction (PCR) at 6 weeks postpartum. During 2005-2012, follow-up of all HIV-infected infants diagnosed in all 13 postnatal care facilities in Francistown, Botswana, was conducted to ascertain patient outcomes. A total of 202 infants were diagnosed with HIV. As of September 2013, 82 (41%) children were alive and on antiretroviral therapy (ART), 79 (39%) had died, and 41 (20%) were either lost to follow-up, had transferred, or their mothers declined ART. Despite success in preventing mother-to-child transmission in Botswana, results of the EID program highlight the need for early diagnosis of HIV-infected infants, prompt initiation of ART, and retention in care.