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For evaluation of genetic diversity among improved alfalfa varieties and Iranian landraces, 12 populations including five improved varieties (Kaysari, Kadi, Ranger, Mesmir, Seariver) and seven landraces (Gharayonje, Amozeynadin, Rahnani, Tazekand, Shazand, Hamedani, Yazdi) were evaluated using agronomic traits and enzyme markers. Thirty-five individuals of each variety were grown and analyzed in separate pots in a unbalanced completely randomized design (CRD). Analysis of variance for agronomic traits showed significant differences for most of the traits among improved and landrace varieties. For esterase and peroxidase enzymes based on presence or absence of enzyme bands (1, 0) eleven polymprphic isozyme bands were detected. For improved and landrace varieties Shanon index mean was 0.48 ± 0.246 and 0.519 ± 0.193, respectively, furthermore Nei genetic diversity index mean for improved and landraces was 0.327 ± 0.181 and 0.352 ± 0.148 respectively, suggesting no difference between improved and landrace varieties was found. Analysis of relation between isozyme markers and agronomic traits showed that there are significant differences between the presence of POX-4 and wet and dry yield in improved varieties.