To determine whether HIV-1 viruses exist in tears of patients under HAART, a cross-sectional study was designed. All participants who underwent long-term HAART with undetectable plasma viral load had detectable HIV-1 viral load in tears (n = 16) and had no difference from the controls (n = 5). Our data suggested that the lacrimal gland and/or other tear-associated tissues could be new reservoirs for HIV-1 and precautions should be taken when doing eye examinations.