Abstract Background Polychlorinated dibenzodioxins (PCDDs), polychlorodibenzofurans (PCDFs), and polychlorobiphenyls (PCBs) are persistent organic pollutants that represent a major concern for women of reproductive age because of the neurodevelopmental effects associated to perinatal exposure. Objectives This study was aimed at characterizing exposure of women of reproductive age to PCDDs, PCDFs, and PCBs as a function of residence in different Italian Regions, in areas at presumable different environmental contamination and human exposure to these pollutants. Methods Study participants were enrolled in 2011–2012 in 6 Italian Regions representative of Northern, Central and Southern Italy; in each region, areas at presumed different exposure (rural, urban and industrial) were selected for enrolment. Each participant provided a serum sample for the analysis of PCDDs, PCDFs and PCBs. Results Median concentrations of PCDDs + PCDFs, DL-PCBs, NDL6-PCBs and NDL9-PCBs in serum samples were respectively 6.0 and 3.5 pgWHO-TE05/g fat, and 75 and 93 ng/g fat. Age was the variable that most affected median serum concentrations. Age adjusted concentrations were found significantly different between geographical zones: women from Northern Italy showed the highest values, followed by Central and Southern Italy. PCDDs + PCDFs concentrations were significantly higher in the group of women residing in industrial areas compared to the group residing in rural areas. A clear diminishing temporal trend was observed compared to levels reported in previous studies. Conclusions This study produced the largest dataset on serum concentrations of PCDDs, PCDFs and PCBs in women of childbearing age in Italy.