The frequency separation temperature characteristics of Nd-doped Dual-frequency Microchip Lasers (DFMLs) were studied. The frequency separation versus the crystal temperature of DFML with different cavity lengths and different kinds of gain medium are experimented. The experimental results show that, the frequency separation is inversely proportional to the cavity length, and positively related to the crystal temperature. The frequency separation change rates with crystal temperatures of 0.5 mm, 0.8 mm and 1 mm DFML (Nd∶YVO4) are 0.34 GHz/℃, 0.12 GHz/℃ and 0.04 GHz/℃, the shorter the cavity length is, the greater frequency separation change with the crystal temperature is. The frequency separation change rate with crystal temperature of 1 mm DFML (Nd∶YVO4) and (Nd∶GdVO4) is nearly same, the simulation agrees with the experimental results well.