The objectives of this study were to (i) assess the extent of genetic variation in soybean microsatellites (simple sequence repeats or SSRs), (ii) assay for amplified sequence length polymorphisms (ASLPs), and (iii) evaluate the usefulness of SSRs and ASLPs as genetic markers. Five microsatellites detected a total of 79 variants (alleles) in a sample of 94 accessions of wild (Glycine soja) and cultivated soybean (G. max). F2 segregation analysis of four of the five microsatellites identified these variants (alleles) with four loci located in independent linkage groups. The number of alleles per microsatellite locus ranged from 5 to 21; to our knowledge these are the largest numbers of alleles for single Mendelian loci reported in soybean. Allelic diversity for the SSR loci was greater in wild than in cultivated soybean. Overall, 43 more SSR alleles were detected in wild than in cultivated soybean. These results indicate that SSRs are the marker of choice, especially for species with low levels of variation as detected by other types of markers. Two alleles were detected at each of the three ASLP loci examined. A total of six ASLP alleles were observed in cultivated soybean and five were observed in wild soybean; all alleles detected in wild soybean were present in cultivated soybean. Allelic diversity values for the ASLP loci were near previous estimates for restriction fragment length polymorphisms and therefore ASLPs may be useful as genetic markers in site-directed mapping.