The objective ot this study was to investigate whether lymphatic markers measured in women during the second trimester are associated with critical congenital heart defects (CCHDs) in offspring. This is a retrospective cohort study of pregnant women who participated in the California Prenatal Screening Program. CCHD data in the offspring was captured by linking birth certificate data with hospital patient discharge records. Second trimester samples were assayed for vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) AA/BB, and PDGF AB. Logistic models were used to evaluate the association between lymphatic biomarkers and CCHD. Models were adjusted for other serum biomarkers and maternal characteristics. Results are presented in odds ratios (OR) with 95% confidence intervals (CI). We identified 93 cases with CCHDs and 194 controls without CCHDs. The crude and adjusted OR for log (ln) VEGF was 1.07 (95%CI 0.94–1.22) and 1.08 (95%CI 0.94–1.24), respectively; for ln PDGF AB/BB was 0.93 (95%CI 0.6–1.35) and 0.58 (95%CI 0.32–1.05), respectively. There was a significant association between ln PDFG AA and CCHDs (crude OR 1.83 (95%CI 1.05–3.2); adjusted OR 2.41 (95%CI 1.06–5.44)). Levels of circulating PDGF AA were highest in cases with hypoplastic left heart syndrome (HLHS) (mean 8.78 +/− 1.54 pg/ml). In this study, increased mid-pregnancy maternal serum levels of PDGF AA were associated with CCHDs in offspring. The highest PDGF AA levels were found in mothers of fetuses with HLHS. These findings may be useful in screening for CCHDs and offer insight into their association with nuchal translucency.