Separations using methanol-water or acetonitrile-water mixtures at different temperatures have been well investigated in reversed-phase liquid chromatography. However, reversed-phase separation with dimethyl sulfoxide (DMSO)-water mixtures is much less studied. In this work, separations of polyhydroxybenzenes, phenol derivatives, benzene, toluene, ethylbenzene, and xylenes (BTEX), and polycyclic aromatic hydrocarbons (PAHs) with DMSO-modified subcritical water were performed at several temperatures to evaluate the effect of temperature on the elution strength of DMSO-water mixtures. The column efficiency obtained by using DMSO-modified subcritical water was also studied. Finally, the resolution of ethylbenzene and p-xylene was investigated.