Abstract: There are two types of alfalfa landraces in Iran. The first is temperate landraces such as Hamedani, Gharah Younjeh and the second is sub-tropical landraces such as Bami, Nikshahri and Yazdi. Do they have different gene pools and could they be identified using agro-morphological traits. A total of 332 landraces which had been collected from different parts of Iran were planted in the experimental field. During 1998-2002, agro-morphological traits were evaluated according to IPGRI descriptors. One way ANOVA analysis showed that the variance among accessions within provinces is not significant for mostly of traits, whereas it was significant among provinces. Cluster analysis revealed that there are five clusters which are grouped in two main clusters. The first main cluster consisted of landraces of central to Northern provinces. The other main cluster consisted of landraces of central to Southern provinces. The results of t-test among quantitative traits in two main clusters and distribution of collected accessions of each province in the produced plot based on two first factor of factor analysis, confirmed the cluster analysis results. Therefore these results revealed that those accessions which were collected from Northern parts of the country were genetically different from Southern accessions. As a result, there are two types of gene pools for alfalfa landraces in Iran. Also this study showed that using agro-morphological traits could be used for distinguishing between two types of gene pools of alfalfa landraces in Iran.