The influence of temperature and gas composition on hydrogen isotope separation factors of membrane cascade with reflux ratio of 0.9998 and different palladium alloy membranes were studied. Separation factors for H-D mixture with different palladium alloy membranes varies from 1.31 to 1.42 at different temperatures, and for H-D-T mixture with Pd-8.6%Y alloy membrane, the factors are 1.2 to 2.0 at different temperatures. The results suggest advantages of operating the cell at high temperature because the higher permeation rate and separation factor at high temperature will lead to a great decrease in the separation stages and membrane area of a separation unit.