Fatty acid ethyl esters (FAEEs) are minor ethanol metabolites that can accumulate in hair. The performance of hair FAEEs as a biomarker that can discriminate null or moderate drinking from risky, excessive drinking was verified by evaluating the relationship between self-reported daily alcohol intake and FAEE concentration in hair. The study subjects were 160 healthy volunteers (52% female) that included teetotallers, moderate/social drinkers (< 60 g of ethanol per day), and heavy drinkers (≥ 60 g/day).The estimated daily alcohol intake (EDAI) was assessed by a specific written questionnaire aimed at estimating the measure and the frequency of alcohol drinking and at excluding confounding factors. FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) were extracted from the hair matrix by overnight incubation in n-hexane/dimethylsulphoxide, purified by solid-phase extraction (SPE) and analyzed by gas chromatography–mass spectrometry (GC-MS) in selected ion monitoring and Electron ionization (EI) mode, using pentadeuterated internal standards. Hair samples exhibited FAEE concentrations (expressed as the sum of the four esters, CFAEE) ranging from 0.01 to 10.78 ng/mg (average 1.16 and median 0.60 ng/mg). The EDAI was from 0 to 246 g of ethanol per day, average 28 g/day and median 15 g/day. A cut-off of 0.5 ng/mg in 3 cm of a proximal hair segment was adopted to discriminate social drinking from excessive ethanol consumption. False positive samples were identified in subjects using ethanol-containing hair lotions and women on estroprogestin therapy. Specificity of 87% was reached when the identified false positives were excluded from data elaboration. CFAEE in hair at a predetermined cut-off can be used to discriminate between moderate and excessive drinking only when confounding factors are meticulously removed. Copyright © 2014 John Wiley & Sons, Ltd.