We estimated mortality rate and predictors of death in children and adolescents who acquired HIV through mother-to-child transmission in Paraguay. In 2000-2014, we conducted a cohort study among children and adolescents aged < 15 years. We abstracted data from medical records and death certificates. We used the Cox proportional hazards model for the multivariable analysis of mortality predictors. A total of 302 subjects were included in the survey; 216 (71.5%) were younger than 5 years, 148 (51.0%) were male, and 214 (70.9%) resided in the Asunción metropolitan area. There were 52 (17.2%) deaths, resulting in an overall mortality rate of 2.06 deaths per 100 person-years. The children and adolescents with hemoglobin levels ≤ 9 g/dL at baseline had a 2-times higher hazard of death compared with those who had levels > 9 g/dL (HR 2.27, 95% CI 1.01-5.10). The mortality of HIV-infected children and adolescents in Paraguay is high, and anemia is associated with mortality. Improving prenatal screening to find cases earlier and improving pediatric follow-up are needed.