Challenges in identifying children exposed prenatally to ethanol necessitate the development of a biomarker for neonates at risk for fetal alcohol spectrum disorder. Meconium fatty acid ethyl esters (FAEE), products of nonoxidative ethanol metabolism, have been established as a novel biomarker of fetal ethanol exposure. We present the first application of this biomarker to a population-based sample in Canada. Six-hundred eighty-two meconium specimens were anonymously collected in the region of Grey Bruce, Ontario, Canada. Meconium FAEE were extracted by liquid-liquid and solid-phase extraction and analyzed by gas chromatography with flame-ionization detection confirmed by gas chromatography with mass spectrometry. We measured ethyl palmitate (E16:0), ethyl palmitoleate (E16:1), ethyl stearate (E18:0), ethyl oleate (E18:1), ethyl linoleate (E18:2), ethyl linolenate (E18:3), and ethyl arachidonate (E20:4). Seventeen of 682 meconium samples tested positive for significant prenatal ethanol exposure (>2.0 nmol/g). FAEE analysis detected fivefold more ethanol-exposed pregnancies than standard postpartum questionnaires in this population (2.5% versus 0.5%) (P < 0.001). The prevalence of ethanol-exposed pregnancies was consistent with Centers for Disease Control and Prevention estimates of "frequent" prenatal drinking and previously published estimates of fetal alcohol spectrum disorder disease prevalence in the general North American population. The FAEE concentrations of negative (95% confidence interval, 0.38-0.49 nmol/g) versus positive (95% confidence interval, 7.74-151.28 nmol/g) samples were distinct, further demonstrating the specificity of this biomarker in determining significant prenatal ethanol exposure. Meconium FAEE analysis demonstrates a fivefold increase in sensitivity over currently used methods of self-report-based screening in Ontario for the detection of ethanol-exposed pregnancies in a clinical setting.