Alfalfa breeding programs have focused to evaluate a large set of genotypes, without considering the ecogeographical and gene pool relationship. Our program is interested to identify prominent germplasm of highly adapted cultivars to central highland of Mexico. For this purpose, we have used molecular markers, lack in M. sativa, and morphological traits to select prominent genotypes, for both grazing and mechanical harvest. Thus, this experimental assay was conducted to develop and characterize 105 SSR markers obtained of genomic libraries and EST and BAC sequence data from the Medicago truncatula and Trifolium pratense genomes into 36 populations. We also included 9 standard cultivars, which represent the historically recognized U.S. germplasm sources. 41 % of the SSR on the M. truncatula genome map amplified polymorphic bands and 25% amplified from the T. pratense, assuming highly conserved SSR and transferable of both legumes to alfalfa populations studied herein. The number of alleles per locus ranged from 3 to 17, with an average of 7.3 and PIC ranged from 0.2 to 0.85, average 0.71. The mean genetic diversity (He) for within and among populations ranged from 0.37 to 0.825, indicating high among-population diversity. A dendogram among 36 populations based on their cluster analysis of GSj (Jaccard coefficient, range 0.32-0.75, mean 0.55) identified four main clusters, representing, 1) fall dormant cultivars (5246, Vernal, Maverick, Hungarian, Ampurdan, Polyhibrida); 2) intermediate (ABI700, Archer, Pierce, Marlborough, Rustique, 15977, Alta Sierra, among others); 3) non-dormant (CUF101, UC-1887&1465, Dona Ana, African pop, Valenciana, Macate, Aragon, Apasco, INIA-76, Puebla-76, Tanverde, Jupiter, San Miguelito, Oaxaca, Florida); 4) other populations representing a wide range of populations world-wide. SSRs were transferable and able to discriminate a number of alfalfa populations. Association mapping in alfalfa is under way to identify genotypes by candidate gene-markers of both T. pratense and M. truncatula.