Abstract: The objective of this study was to characterize and classify the genetic diversity among alfalfa (Medicago sativa L.) ecotypes collected from the cold regions of Iran, based on some agro-morphological traits. Twenty one alfalfa ecotypes were collected and planted in a Randomized Complete Blocks Design (RCBD) with three replications in April 1998 at Nyshabour Agricultural and Natural Resource Research Station, Khorasan Razavi, Iran. Twenty three above ground agro-morphological characters were recorded during the growing seasons of 1999-2001. The variables were analyzed by descriptive statistics and multivariate statistical procedures to discriminate differences among genotypes and determine groups based on their similarities. Factor analysis was performed for all agro-morphological traits and reduced them down to 6 common factors which accounted for 80.45% of total variations among the genotypes studied. The twenty one ecotypes were classified in to 4 clusters by cluster analysis. Each group had at least one trait which made it different from the other groups (group 1: No. of pods per raceme and 100-seed weight; group 2: forage yield, dry matter yield, regrowth rate and stem dry matter yield; group 3: leaf-stem ratio, leaf dry matter yield and group 4: seed yield). These results suggest the presence of variation among alfalfa ecotypes available in cold regions in Iran, which could be considered for further breeding strategies and studies.