Abstract: The study aimed to investigate the herbicide atrazine tolerance characteristics of 37 alfalfa varieties at the germination stage, analyse the growth and development characteristics of different alfalfa under atrazine stress and establish an evaluation system for its screening, and select alfalfa germplasms tolerant to atrazine at the germination stage. The atrazine tolerance measure（A value）, average affiliation function value（AFV value） and weighted correlation coefficient（WAC value） were combined with correlation analysis, principal component analysis and cluster analysis to identify the atrazine tolerance of alfalfa materials at the germination stage. The results showed that root length, dry weight and SPAD of 37 alfalfa varieties were more severely inhibited under the stress of 21 mg/L atrazine solution concentration Base on A value, AFV value and WAC value comprehensive rankings, the varieties more tolerant to atrazine were Fortress, Philly and Reindeer. The A value clustering analysis classified the tested varieties into four tolerance classes, with only one variety of Fortress in Class Ⅰ, 13 varieties in Class Ⅱ, 9 varieties in Class Ⅲ and 14 varieties in Class Ⅳ. The most atrazine-tolerant varieties were identified as Fortress, followed by Philly, Dite and Reindeer, which could be used as important germplasm resources for breeding and atrazine-tolerance research in alfalfa.