BACKGROUND: The intergenerational effects of HIV require long-term investigation. We compared developmental outcomes of different generations impacted by HIV-children of mothers not living with HIV, the 'second generation' (ie, with recently infected mothers) and the 'third generation' (ie, children of perinatally infected mothers). METHODS: A cross-sectional community sample of N=1015 young mothers (12-25 years) and their first children (2-68 months, 48.2% female), from South Africa's Eastern Cape Province. 71.3% (n=724) of children were born to mothers not living with HIV; 2.7% (n=27; 1 living with HIV) were third-generation and 26.0% (n=264; 11 living with HIV) second-generation children. Child scores on the Mullen Scales of Early Learning (MSEL), the WHO Ten Questions Screen for Disability and maternal demographics were compared between groups using χ2 tests and univariate approach, analysis of variance analysis. Hierarchical linear regressions investigated predictive effects of familial HIV infection patterns on child MSEL composite scores, controlling for demographic and family environment variables. RESULTS: Second-generation children performed poorer on gross (M=47.0, SD=13.1) and fine motor functioning (M=41.4, SD=15.2) and the MSEL composite score (M=90.6, SD=23.0) than children with non-infected mothers (gross motor: M=50.4, SD=12.3; fine motor: M=44.4, SD=14.1; composite score: M=94.1, SD=20.7). The third generation performed at similar levels to non-exposed children (gross motor: M=52.4, SD=16.1; fine motor: M=44.3, SD=16.1, composite score: M=94.7, SD=22.2), though analyses were underpowered for definite conclusions. Hierarchical regression analyses suggest marginal predictive effects of being second-generation child compared with having a mother not living with HIV (B=-3.3, 95% CI=-6.8 to 0 .1) on MSEL total scores, and non-significant predictive effects of being a third-generation child (B=1.1, 5% CI=-7.5 to 9.7) when controlling for covariates. No group differences were found for disability rates (26.9% third generation, 27.7% second generation, 26.2% non-exposed; χ2=0.02, p=0.90). CONCLUSION: Recently infected mothers and their children may struggle due to the disruptiveness of new HIV diagnoses and incomplete access to care/support during pregnancy and early motherhood. Long-standing familial HIV infection may facilitate care pathways and coping, explaining similar cognitive development among not exposed and third-generation children. Targeted intervention and fast-tracking into services may improve maternal mental health and socioeconomic support.