Hypospadias is a congenital malformation of penile urethra with unknown etiology in most cases. Persistent organic pollutant (POP) exposure may disrupt endocrine function during a critical window of development of male genitalia. In animal studies, POPs have been associated with male reproductive disorders, including hypospadias, but only few studies have assessed this relationship in humans. The aim of this study is to inves-tigate the association between hypospadias and POP concentration levels in breast milk, as a proxy for prenatal exposure. This is a nested case-control study of Danish and Finnish mother-son pairs. Maternal breast milk samples were collected between 1997 and 2002, and they represent infant boys born with hypospadias [n = 33 (n = 22 Danish and n = 11 Finnish)] and their 1:1 matched controls. Breast milk samples were analyzed for six classes of POPs [including dioxins, polychlorinated biphenyls, flame retardants and perfluorinated alkylated substances (PFAS)]. We estimated odds ratios (ORs) and 95% confidence intervals (CI) for each chemical class using conditional logistic regression. In addition, a composite exposure score system was used to explore the effect of a POP mixture (four chemical classes): The composite score was categorized as low, moderate, or high exposure, and differences between cases and controls were tested with conditional logistic regression. No sta-tistically significant associations were observed between the sums of the chemical classes and hypospadias in either country. The composite score was unable to detect differences in the risk of hypospadias between the tertiles of POP exposure. Levels of PFAS were significantly higher in Danish than in Finnish breast milk samples. This small study does not provide evidence for an association between hypospadias and exposure to POPs but adds information on quantitative exposures. Further development of multi-exposure models is needed for assessing the potential mixture effect associated with multiple chemical exposures.