Exposure to mercury, a risk factor for neurodevelopmental toxicity, was assessed in Spanish children (preschool children and newborns, n = 218) in a four-locations survey by performing mercury determination in hair. To assess the prenatal and children's exposure and its potential risk, total mercury (THg) and methylmercury (MeHg) were analyzed and examined for associations with maternal sociodemographic characteristics and dietary intake through interviews and food frequency questionnaires. The mean THg in hair was 0.94 microg/g, ranging from 0.19 to 5.63 microg/g in preschool children and 1.68 microg/g (0.13-8.43 microg/g) in newborns. Associations between mercury levels in hair and fish consumption frequency were found regardless of the group evaluated. Neither other food item nor maternal covariates were associated with mercury levels in the newborn group. In children, the mean THg values among frequent fish consumers (more than four times per week) were almost threefold higher compared with non-consumers (1.40 vs. 0.49 microg/g). Newborns from mothers who had intake of fish two or more times per week exhibited nearly threefold higher hair levels than those who rarely or never consumed fish (2.26 vs. 0.78 microg/g). Finally, mercury levels in hair exceeded the EPA reference dose (RfD) of 0.1 microg Hg/kg body weight per day (equivalent to 1 microg Hg/g hair) in 42% of the population studied. Overall, we conclude that levels of mercury in Spain are among the highest in the non-exposed populations probably because of the relatively high fish consumption.