Crop reduction caused by cryogenesis and drought is a serious and global problem. The environmental stress caused by low temperature and drought during the overwintering stage of forage is the key factor leading to this low yield. In cold and arid grassland, winter irrigation can effectively alleviate the stress of alfalfa during overwintering, improve the survival rate of alfalfa, and significantly increase the yield. However, the water uptake patterns of alfalfa under winter irrigation are not clear, which are important to explore the mechanism of alleviating environmental stress by winter irrigation. In this research, the stable isotope compositions of all probable water sources and alfalfa xylem water were measured after winter irrigation. A graphical method was applied to identify the main soil layers with water uptake by the alfalfa roots. The contribution rate of available water sources to alfalfa xylem water was quantified by the MixSIAR (Bayesian isotope analysis mixing model in R) model. The results indicated that alfalfa absorbed soil water when the soil water content was high enough in the root layer when under high water volume freezing irrigation (irrigation in early winter when soil is freezing) but not under low and medium water volume freezing irrigation. Alfalfa gradually began to absorb soil water on the third day after thawing irrigation (irrigation in late winter when the soil is thawing) and showed different water uptake characteristics under low, medium, and high water volume. Thawing irrigation also accelerated the regeneration of alfalfa.