The level of variation of simple sequence repeat (SSR) markers in cultivated alfalfa from American, Australian and Chinese sources was evaluated using a novel autotetraploid statistical method to calculate the effective number of alleles, the allele frequencies and heterozygosity. We used 19 SSR primers to screen seven polymorphic SSR loci in 320 plants from eight populations. The genetic distance and phylogenetic analysis (DISPAN) program was used to calculate the inter- and intra-population genetic relationships using the conventional binary absence/presence (0/1) method and our novel autotetraploid method. The autotetraploid method resulted in significantly higher heterozygosity (p < 0.01), average effective number (p < 0.01) and lower standard genetic distance (p < 0.01) than the binary method. Our results suggest that our new autotetraploid method is a very useful tool for assessing genetic variation and genetic relationships in all autotetraploid plant species.