Abstract We explore the influence that polymer molecular weight, total polymer concentration and temperature have on the interfacial tension of aqueous two-phase systems. We report the interfacial tension at 4°C, 25°C and 40°C for sixteen different PEG/Dx/water systems which consist of all possible combinations of the four PEG molecular weights 4,000, 6,000, 10,000 and 20,000 and the four Dx molecular weights 10,000, 40,000, 110,000 and 500,000. Four tie lines were measured for each system. We show that there exists a good linear correlation between the log of the interfacial tension and log of the tie line length, the difference in Dx concentration between the top and bottom phase ( ΔDx ≡ [ Dx ] T − [ Dx ] B ) and the difference in PEG concentration between the top and bottom phase ( ΔPEG ≡ [ PEG ] T − [ PEG ] B ). The linear correlation between the log of the interfacial tension and ΔDx or ΔPEG suggests the possibility of using interfacial tension as an analytical tool to measure phase composition. When systems of the same tie line length are compared, increasing the molecular weight of one of the polymers increases the interfacial tension. We find that systems closer to the critical point (short tie line length) are more sensitive to changes in polymer molecular weight than systems far away from the critical point (long tie line length). The trend of interfacial tension with temperature is less clear than the trend with polymer molecular weight. The double log curves of interfacial tension versus tie line length at different temperatures cross each other in such a way that an increase in the temperature will increase, decrease or not affect the interfacial tension depending on the tie line length that we consider.