Accurate drug use identification through subjective self-report and toxicological biosample (hair) analysis are necessary to determine substance use sequelae in youth. Yet consistency between self-reported substance use and robust, toxicological analysis in a large sample of youth is understudied. We aim to assess concordance between self-reported substance use and hair toxicological analysis in community-based adolescents. Hair results by LC-MS/MS and GC-MS/MS and self-reported past-year substance use from an Adolescent Brain Cognitive Development (ABCD) Study subsample ( = 1,390; ages 9-13; 48% female) were compared. The participants were selected for hair selection through two methods: high scores on a substance risk algorithm selected 93%; 7% were low-risk, randomly selected participants. Kappa coefficients the examined concordance between self-report and hair results. 10% of youth self-reported any past-year substance use (e.g. alcohol, cannabis, nicotine, and opiates), while a mostly non-overlapping 10% had hair results indicating recent substance use (cannabis, alcohol, non-prescription amphetamines, cocaine, nicotine, opiates, and fentanyl). In randomly selected low-risk cases, 7% were confirmed positive in hair. Combining methods, 19% of the sample self-reported substance use and/or had a positive hair sample. Kappa coefficient of concordance between self-report and hair results was low (kappa = 0.07;  = .007). Hair toxicology identified substance use in high-risk and low-risk ABCD cohort subsamples. Given low concordance between hair results and self-report, reliance on either method alone would incorrectly categorize 9% as non-users. Multiple methods for characterizing substance use history in youth improves accuracy. Larger representative samples are needed to assess the prevalence of substance use in youth.