Asparagus is a large genus comprising three subgenera (Asparagus, Protasparagus and Myrsiphyllum) with species distributed in Africa, Asia, Australia and Europe. The southwest of Asia includes the natural distribution of several species of this genus. Also, the proposed center of origin of garden asparagus (A. officinalis) is located in this region. The genetic diversity and the phylogenetic relationships among twelve diploid and polyploid (4x, 8x and 10x) Asparagus accessions (7 A. officinalis + 3 A. verticillatus + 1 A. persicus + 1 A. breslerianus), collected from different sites of Iran, were assessed in this study. Around ten plants per accession were analyzed using a set of six expressed sequence tag-derived simple sequence repeats (EST-SSRs) primer pairs. An average of 23 alleles per marker was detected in the studied collection, and an octoploid A. officinalis showed the highest number of alleles (62 alleles total). A cluster analysis was carried out with the alleles matrix (1 = presence, 0 = absence). In the dendrogram obtained, all individuals from each accession were grouped together and the different accessions were clearly separated. In addition, three main groups were obtained: a first group was formed by both A. verticillatus and A. persicus populations, a second one by diploid A. officinalis, and the third group joined the polyploid A. officinalis (4x, 8x and 10x) and the octoploid A. breslerianus. A high genetic variability was observed in this study, which could be used to broaden the genetic base of garden asparagus.