Abstract: The research aimed to explore the root tip squash technique of Alfalfa and provide references for the chromosome technique. With root tips of Alfalfa as tested materials, which test the pretreatment solution, pretreatment time, fixed-time, staining and staining time. The results showed that, 8-hydroxyquinoline solution for the pretreatment, pretreatment 2.5 h, fixed with carnoy’s fixative 20 h, with a 1 mol/L HCl solution at 60℃, from 8 min, using 45% acetic at room temperature to soften 20 min, stained with Giemsa for the dye 3 h, which was a better way to technical chromosome of Alfalfa. The research provided a new method for the direct identification of polyploid Alfalfa.