Since 2012, the WHO recommends Option B+ for the prevention of mother-to-child transmission of HIV. This approach entails the initiation of lifelong antiretroviral therapy in all HIV-positive pregnant women, also implying protection during breastfeeding for 12 months or longer. Research on long-term adherence to Option B+ throughout breastfeeding is scarce to date. Therefore, we conducted a prospective observational cohort study in Fort Portal, Western Uganda, to assess adherence to Option B+ until 18 months postpartum. In 2013, we recruited 67 HIV-positive, Option B+ enrolled women six weeks after giving birth and scheduled them for follow-up study visits after six, twelve and 18 months. Two adherence measures, self-reported drug intake and amount of drug refill visits, were combined to define adherence, and were assessed together with feeding information at all study visits. At six months postpartum, 51% of the enrolled women were considered to be adherent. Until twelve and 18 months postpartum, adherence for the respective follow-up interval decreased to 19% and 20.5% respectively. No woman was completely adherent until 18 months. At the same time, 76.5% of the women breastfed for ≥12 months. Drug adherence was associated with younger age (p<0.01), lower travel costs (p = 0.02), and lower number of previous deliveries (p = 0.04). Long-term adherence to Option B+ seems to be challenging. Considering that in our cohort, prolonged breastfeeding until ≥12 months was widely applied while postpartum adherence until the end of breastfeeding was poor, a potential risk of postpartum vertical transmission needs to be taken seriously into account for Option B+ implementation.