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Objectives: Mother-child pairs may separate during early life, yet the health impacts thereof are unclear. We explored the patterns and impact of separation among women living with HIV (WLHIV) and their children in South Africa., Methods: WLHIV who had initiated antiretroviral therapy (ART) during pregnancy received HIV viral load (VL) testing and completed a timeline questionnaire of mother-child separation since delivery at 3-5 years post-partum. Health care usage was abstracted from routine medical records. We examined associations between separation and (a) maternal health outcomes (engagement in HIV care and HIV viral suppression, [VS]) and (b) child health outcomes (post-breastfeeding HIV testing and immunisation completion), using logistic regression., Results: Of 346 mother-child pairs (median maternal age at antenatal ART initiation, 28 years), 24% were ever separated (median time to first separation 20 months, interquartile range [IQR] 9, 31). Most separated children were living with their grandmothers (65/83, 78%). Mothers who ever separated were younger, and more likely to be employed, and to reside in informal housing than those who never separated. Any separation reduced the odds of VS ≤ 50 copies/mL at four years post-partum (odds ratio 0.57; 95% CI 0.34-0.93); associations were similar for VL ≤ 1000 copies/mL and maternal engagement in care. No association was found between separation and child confirmatory HIV testing or immunisation completion., Conclusions: In this setting, mother-child separation is common in the first four years of life and appears associated with suboptimal maternal outcomes. Further research is required to understand the drivers and implications of mother-child separation., (© 2020 John Wiley & Sons Ltd.)