Dioxins, polychlorinated biphenyls (PCBs), and organochlorine pesticides are bioaccumulative chemicals that are considered to be toxic contaminants. These chemicals are classified as persistent organic pollutants (POPs). We have started a prospective cohort study to examine the effects of perinatal exposure to these chemicals as well as methylmercury on neurobehavioral development in Japanese children. In this article, the method and the consequence of exposure assessment were described. The concentrations of POPs in maternal blood, cord blood, and breast milk were determined by high-resolution gas chromatography–high-resolution mass spectrometry. In breast milk samples, p,p ′-DDE was the predominant pollutant, total PCB and β-HCH being the other major constituents. Mirex and major toxaphenes were also detected in all samples even though these chemicals have never been used in Japan. Simple correlation analysis showed high correlations among the three sample materials, indicating that there was a high degree of consistency of chemicals in the body. Major chemicals were also intercorrelated with other chemicals in either of the three sample materials. These findings indicate the presence of coexposure to multiple POPs. Multiple regression analysis indicated that the concentrations of PCBs were affected by the age of mother, parity, and maternal fish intake. These results are informative in terms of considering the strategy to reduce the body burden of POPs in females. The chemical analysis were performed by two different institutes. Multiple regression analysis also showed that the result of chamical analysis was affected significantly by the difference in analyzing institute. This finding suggest the importance of consistency in chemical analysis and the necessity for quality and accuracy control using reference materials.