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Objective: World Health Organization recommends promoting breastfeeding without restricting its duration among HIV-positive women on lifelong antiretroviral treatment (ART). There is little data on breastfeeding duration and mother to child transmission (MTCT) beyond 24 months. We compared the duration of breastfeeding in HIV-exposed and HIV-unexposed children and we identified factors associated with postpartum-MTCT in a semi-rural population of Mozambique. Methods: This cross-sectional assessment was conducted from October-2017 to April-2018. Mothers who had given birth within the previous 48-months in the Manhiça district were randomly selected to be surveyed and to receive an HIV-test along with their children. Postpartum MTCT was defined as children with an initial HIV positive result beyond 6 weeks of life who initiated breastfeeding if they had a first negative PCR result during the first 6 weeks of life or whose mother had an estimated date of infection after the child's birth. Cumulative incidence accounting for right-censoring was used to compare breastfeeding duration in HIV-exposed and unexposed children. Fine-Gray regression was used to assess factors associated with postpartum-MTCT. Results: Among the 5000 mother-child pairs selected, 69.7% (3486/5000) were located and enrolled. Among those, 27.7% (967/3486) children were HIV-exposed, 62.2% (2169/3486) were HIV-unexposed and for 10.0% (350/3486) HIV-exposure was unknown. Median duration of breastfeeding was 13.0 (95%CI:12.0–14.0) and 20.0 (95%CI:19.0–20.0) months among HIV-exposed and HIV-unexposed children, respectively (p<0.001). Of the 967 HIV-exposed children, 5.3% (51/967) were HIV-positive at the time of the survey. We estimated that 27.5% (14/51) of the MTCT occurred during pregnancy and delivery, 49.0% (2551) postpartum-MTCT and the period of MTCT remained unknown for 23.5% (12/51) of children. In multivariable analysis, mothers' ART initiation after the date of childbirth was associated (aSHR:9.39 [95%CI:1.75–50.31], p = 0.001), however breastfeeding duration was not associated with postpartum-MTCT (aSHR:0.99 [95%CI:0.96–1.03], p = 0.707). Conclusion: The risk for postpartum MTCT was nearly tenfold higher in women newly diagnosed and/or initiating ART postpartum. This highlights the importance of sustained HIV screening and prompt ART initiation in postpartum women in Sub-Saharan African countries. Under conditions where HIV-exposed infants born to mothers on ART receive adequate PMTCT, extending breastfeeding duration may be recommended. [ABSTRACT FROM AUTHOR]