Genetic diversity within and between 30 ecotypes of alfalfa (Medicago sativa L.) collected from North West of Iran was analysed at the DNA level by RAPD technique. DNA extraction was performed individually for 30 seedlings as well as bulked samples of 30 ecotypes. Using 10 arbitrary primers, 78 DNA fragments were scored and were varied between 36 to 68 bands. Varzeghan – Joshin and Tabriz- Sparakhon ecotypes showed the highest and the lowest genetic diversities, respectively. Marand-Zounragh with Bostanabad- Bashkand and Varzeghan-Joshin with Varzeghan-Alhord showed the highest genetic distance, respectively. The highest and the lowest genetic distances were belonged to Marand-Zounoragh with Bostanabad- Bashkand and Varzeghan-Joshin with Varzeghan- Alhord ecotypes, respectively. This result was in agreement with geographic distance of ecotypes. The results of molecular variance analyses showed 65.37% and 34.63% genetic diversity between and within ecotypes, respectively. This can be used for ecotype selection and improvement. Cluster analyses based on UPGMA algorithm, grouped 30 ecotypes into 4 clusters. Bostanabad- Bashkand ecotype was individually grouped in a separate cluster. The growth location of this ecotype was different from others, as it was located in a very cold region. Hence, might be cold tolerant ecotype. This may suggest that the altitude level of regions influences the ecotypes genetic structure much more than the environmental factors.