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Alfalfa (Medicago sativa), known as green gold, is one of the best-known forage plants in the world. Alfalfa is an autotetraploid allogamous plant with a large genetic diversity among its cultivars. This diversity is due to self-incompatibility, a high rate of allogamy, and open pollination. This study aimed to investigate the biodiversity of grain storage proteins of M. sativa using gel electrophoresis. For this purpose, seed proteins of 12 Iranian genotypes including seeds collected from Lenjan of Isfahan, Dashte Qazvin, Asadabad, Qasr Shirin, Taleghan Qazvin, Malayer, Isfahan, Saqez, Gonbad Kavous, Kamyaran, Shiraz, and Najafabad of Isfahan were studied and a total of 22 strips were identified. Then, the strips were used to estimate the genetic distance of alfalfa genotypes. The results of the study based on the presence and absence of specific protein bands in the seed protein pattern showed that genotypes 1 (Collection from Isfahan-Lenjan) and 12 (Collection from Najafabad-Isfahan) have the most variation. A similarity matrix was calculated and cluster analysis was performed based on the Jaccard similarity coefficient by the UPGMA method. The similarity coefficients ranged from 0.3 to 1. In general, classification based on cluster analysis showed a similar trend to the differences observed in gel electrophoresis. The total protein of population number 12 (Najafabad population of Isfahan) had the highest amount among the studied genotypes. The highest and lowest number of bands were related to genotype number 12 (population of Najafabad, Isfahan) and genotype number 11 (population collected from Shiraz), respectively. Therefore, the results showed that the protein pattern of alfalfa seeds based on the presence and absence of protein bands can determine genetic diversity. These findings can be used in future research in the field of proteomics and breeding studies in future.