Substance misuse during pregnancy can result in a variety of poor pregnancy outcomes. Objective data reporting the prevalence of neonates born with ethanol metabolites (evidence of prenatal ethanol exposure) in their fluids or tissues are limited. A secondary analysis of umbilical cord tissue specimens received for routine toxicological analysis was conducted. Prevalences of ethyl glucuronide (EtG), a long-term direct ethanol biomarker, were determined using a new laboratory tool, LDTD-MSMS. Additionally, other commonly misused substances were determined using routine procedures. Of 12,995 specimens, 238 (1.8%) specimens contained EtG. Concentrations of EtG ranged from 5 ng/g to 6679 ng/g (median 47 ng/mg; IQR: 16 ng/g, 203 ng/g). Of those 238 EtG-positive specimens, nearly 58% (N = 138) contained additional substances or metabolites. Self-report of substance use during pregnancy is under-reported. We have demonstrated co-exposure of substances with ethanol is higher than previous reports.