Background Hair antiretroviral concentration has served as an innovative and objective measure of antiretroviral adherence. However, some factors (for example, pharma-cokinetics and hair characteristics) may contribute to the variability of hair antiretroviral concentration that may threaten the validity and reliability of the hair measure as a biomarker of adherence. This study aimed to examine the potential factors that may influence the measure of hair antiretroviral concentration. Methods Hair samples from a cohort of 372 people living with HIV (PLHIV) receiving lamivudine (300 mg/day) in Guangxi, China. Lamivudine concentration was analysed using liquid chromatography-tandem mass spectrometry. Multivariable linear regression was used to evaluate the associations of hair lamivudine concentration with age, sex, ethnicity, height, weight, body mass index, duration of HIV diagnosis, duration of current regimen, dosing schedule, concomitant antiretroviral medications, frequency of hair washing, hair care products use, hair cosmetic treatment and self-reported adherence. Results Multivariable models revealed that frequency of hair washing (β=-0.221, P=0.001), dosing schedule (β=0.141, P=0.036) and self-reported adherence (β=0.160, P=0.002) were associated with hair lamivudine concentration. Conclusions We observed that, among those potential factors, hair lamivudine concentration was influenced by frequency of hair washing and dosing schedule. Therefore, frequency of hair washing and dosing schedule should be considered in future research using hair lamivudine concentration as a measure of lamivudine exposure and bio-marker of adherence.