Background: We sought to compare self-reported alcohol consumption using Timeline Followback (TLFB) to biomarker-based evidence of significant alcohol use (phosphatidylethanol [PEth] > 20 ng/ ml). Using data from patients with HIV (PWH) entering a clinical trial, we asked whether TLFB could predict PEth > 20 ng/ml and assessed the magnitude of association between TLFB and PEth level. Methods: We defined unhealthy alcohol use as any alcohol use in the presence of liver disease, atrisk drinking, or alcohol use disorder. Self-reported alcohol use obtained from TLFB interview was assessed as mean number of drinks/day and number of heavy drinking days over the past 21 days. Dried blood spot samples for PEth were collected at the interview. We used logistic regression to predict PEth > 20 ng/ml and Spearman correlation to quantify the association with PEth, both as a function of TLFB. Results: Among 282 individuals (99% men) in the analytic sample, approximately two-thirds (69%) of individuals had PEth > 20 ng/ml. The proportion with PEth > 20 ng/ml increased with increasing levels of self-reported alcohol use; of the 190 patients with either at-risk drinking or alcohol use disorder based on self-report, 82% had PEth > 20 ng/ml. Discrimination was better with number of drinks per day than heavy drinking days (AUC: 0.80 [95% CI: 0.74 to 0.85] vs. 0.74 [95% CI: 0.68 to 0.80]). The number of drinks per day and PEth were significantly and positively correlated across all levels of alcohol use (Spearman's R ranged from 0.29 to 0.56, all p values < 0.01). Conclusions: In this sample of PWH entering a clinical trial, mean numbers of drinks per day discriminated individuals with evidence of significant alcohol use by PEth. PEth complements self-report to improve identification of self-reported unhealthy alcohol use among PWH.