A series of new 5-alkyl-2-benzylsulfanylpyrimidin-4(3H)-ones (5a–y) bearing different substituted arylmethyl moieties at the C-6 position of the pyrimidine core have been synthesized and evaluated for their in vitro activities against HIV-1 and HIV-2 in MT-4 cell cultures. The majority of the title compounds showed moderate to good activities against HIV-1 with an IC 50 range from 6.67μM to 0.12μM. Among them, 6-(3,5-dimethylbenzyl) analogue 5q exhibited the most potent anti-HIV-1 activity (IC 50 =0.12μM, SI>2642), which was about 40-fold more active than the reference compounds 1-[(2-hydroxyethoxy)methyl]-6-(phenylsulfanyl)thymine (HEPT) and 2′,3′-dideoxyinosine (DDI). The structure–activity relationships (SARs) of these new congeners were further discussed.