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During pregnancy, women are commonly exposed to several endocrine-disrupting chemicals, including persistent organic pollutants (POPs). These compounds can transfer to the fetus through the placenta. Prenatal POP exposure is related to altered fetal genital and reproductive tract development. However, the relationship between exposure to POP mixtures and anogenital distance (AGD) is poorly investigated. This study investigated the association between prenatal exposure to POP mixtures and AGD in 8-year-old children. Data were collected from the INMA-Asturias cohort. Maternal serum POP concentrations were measured during the first trimester of pregnancy. Anoscrotal distance (AGD AS) and anopenile distance (AGD AP) in males and anofourchetal distance (AGD AF) and anoclitoral distance (AGD AC) in females were recorded in 362 8-years-olds. Conventional linear regression, and the novel weighted quantile sum regression (WQSR) and Bayesian kernel machine regression (BKMR) models were applied to assess the relationships between AGD and POPs exposure stratified by sex. Among males, in the linear regression, b-hexachlorocyclohexane, PCB138, PCB153, and PCB180 were inversely associated with the anogenital index (AGI) AS (−0.06 mm/kg (95% confidence interval [CI]: –0.11, −0.02), −0.07 mm/kg (95% CI: –0.14, −0.01), −0.07 mm/kg (95% CI: –0.13, −0.01), and −0.08 mm/kg (95% CI: –0.14, −0.02), respectively). Among females, polybrominated diphenyl ether (PBDE)47 and PBDE154 were positively associated with increased AGI AF (0.02 mm/kg (95% CI: 0.00, 0.03) and 0.09 mm/kg (95% CI: 0.01, 0.17), respectively). BKMR confirmed these associations. WQSR found a negative combined effect of the POP mixture on AGD, and PCB138, PCB153, and PCB180 (weighted 0.18, 0.13, and 0.09, respectively) were identified as the most impacting chemicals. In females, WQSR found a positive combined effect and determined PBDE47 (weighted 0.35) as the most impacting. Maternal exposure to a POP mixture was negatively associated with AGD in male children and positively associated with AGD in female children, thus providing evidence of the adverse effects of POPs on genital development. • The joint effect of 18 POPs exposure on AGD was assessed through linear regression, WQSR and BKMR statistical approaches. • Co-exposure to POPs is related to a reduced AGD in male children and to an increase of AGD in female children at 8 years. • Exposure to POPs with anti-androgenic effects during pregnancy may hamper normal masculinization of the reproductive system. [ABSTRACT FROM AUTHOR]