The interpass temperature plays a decisive role in the welding quality of high strength low alloy steel. Especially for multilayer multi-pass welds,the thermal cycle of weld interacts with each other,and the phase transformation is extremely complex. The effect of interpass temperature on the microstructure and cold crack of HAZ of high strength low alloy steel 16MND5 is systematically studied. The experiments are carried out at the room temperature of 125 ℃,250 ℃ and 400 ℃ respectively. The result show that cold cracks occur only in the coarse grained zone of HAZ（CGHAZ） at room temperature,and the corresponding hardness of CGHAZ is the highest（up to 400 HV）. The CGHAZ matrix structures corresponding to the four interlayer temperatures are incompletely tempered martensite,tempered martensite,tempered martensite and lower bainite,and granular bainite respectively. In addition,the widmanstatten appears near the fusion line in CGHAZ obtained from all the interpass temperatures except room temperature,especially at 250 ℃. The preheating temperature of 125～250 ℃ is regarded as the most suitable according to the hardness,microstructure and fracture of welded joints.