We examined the association between maternal characteristics, routinely collected first- and second-trimester biomarkers and the risk of having an infant with a critical congenital heart defect (CCHD). Included were women who participated in the California Prenatal Screening Program who had nuchal translucency (NT) measurement and first- and second-trimester serum screening. All pregnancies ended in a live birth of an infant without aneuploidy or a neural tube defect. Poisson regression analyses were used to estimate the relative risk and 95% confidence interval of a CCHD by maternal characteristics, first- and second-trimester serum biomarkers or NT measurements. The sample included 118,194 mother-infant pairs; 284 infants had a CCHD. Women with preexisting diabetes were three-times as likely to have an infant with a CCHD. After adjusting for preexisting diabetes, women with first-trimester human chorionic gonatotropin (hCG) measurement <10th centile were 1.6-times as likely to have an infant with a CCHD (P = 0.011). Women with a NT measurement ≥95th centile were at two- to threefold higher risk of having an infant with a CCHD (P's = 0.004-0.007). Pregnancies with two risk factors for an infant with a CCHD were 5.6-times more likely to have an infant with a CCHD than women with no identified risk factors (P < 0.001). Despite the increased risk, performance testing demonstrated low sensitivity and specificity for screening use of these risk factors. Of the women with an infant with a CCHD, only 21.8% had an identified risk factor. © 2016 Wiley Periodicals, Inc.