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Analysis and dissection of native germplasm resources and characterization of their relationships are essential for conservation and plant breeding programs. To study the diversity pattern of alfalfa populations in the East Azerbaijan province of Iran, 900 accessions belonging to 30 local populations were collected and subjected to microsatellite profiling. Seven simple sequence repeat (SSR) loci were harnessed to analyze and characterize the studied populations. Data driven from microsatellite markers were analyzed, and diversity indices such as the effective number of alleles, polymorphic information content (PIC), and Shannon's index underwent estimation. Moreover, cluster analysis using the neighbor-joining algorithm and principal coordinate analysis were further performed to elucidate the relationships among the studied populations. Totally, 47 SSR alleles were observed in the studied alfalfa populations. The mean allele numbers per locus were 6.71 and ranged from 2 to 12. Additionally, the PIC for the applied SSRs was 0.66. The analysis of molecular variance indicated a significant difference between and within the studied alfalfa populations (P < 0.00001). Furthermore, genetic distance analysis demonstrated that populations are clustered into three distinct groups, of which Ghara-Yonjeh had a larger distance from other populations. These results revealed that the studied local population in the northwest regions of Iran harbors higher genetic diversity and is a valuable resource for future alfalfa germplasm conservation and breeding programs. [ABSTRACT FROM AUTHOR]