Despite a dramatic decrease in vertical transmission of HIV in the developed world, maternal HIV infection acquired after negative prenatal screening still leaves a window of vulnerability. Through quality assurance programs in two Canadian Provinces, five cases where perinatal HIV transmission occurred despite negative prenatal screening were identified between 2005 and 2015. Maternal risk factors such as intravenous drug use, high-risk sexual behavior, hepatitis C virus co-infection, and belonging to high prevalence minority groups were common. Two mothers had their negative HIV test performed in the first trimester and three mothers had negative testing in the third trimester. All babies were clinically healthy at delivery with a normal weight. Three babies were tested following subsequent identification of maternal HIV infection and two babies presented with opportunistic infections leading to their diagnoses. The characteristics of these cases suggest that to achieve complete elimination of vertical HIV transmission, selective and innovative clinical management of mothers at high risk for HIV may be required.