To gain a better understanding of wild and weedy azuki population structures in relation to the cultigens we have developed simple sequence repeat (SSR) markers based on a new methodology for plant material. In the azuki bean genome, the number of (AG)(n) and (AC)(n) motif loci per haploid genome has been estimated to be 3,500 and 2,100, respectively, indicating that (AG)(n) motifs are a rich source of markers. We constructed a (AG)(n)-SSR-enriched library in azuki bean in order to obtain a comprehensive range of SSR markers efficiently. The method applied in this study resulted in a 116-fold enrichment over the non-enriched genomic library, with a high percentage (98%) of successful single-locus amplification by the primer pairs designed. Consequently, this method can be applied to construct SSR-enriched libraries suitable for large-scale sequencing. We obtained 255 unique sequences from an (AG)(n)-enriched library for azuki bean. Fifty primer pairs were designed and screened against five populations of wild azuki bean. Among these five populations, one population from Bato town, Tochigi prefecture, Japan, showed greater polymorphism using these primers than the others and was therefore chosen for the in-depth study. The genotypes of 20 individuals were investigated using eight of the SSR primers developed. The genetic relationships among individuals revealed a complex spatial pattern of population structure. Although azuki bean is considered to be a predominantly self-pollinating species, 3 of the 20 individuals tested in the population showed heterozygous genotypes, indicating outcrossing. Allele size and DNA sequence in each of the 20 individuals were compared with those of landraces and released cultivars of azuki bean. Plants in part of the population had many alleles of the same size and with the same sequence as those in cultivated azuki bean, suggesting that gene flow from the cultigen to wild plants has occurred in this population. Unintentional transgene escape from azuki could therefore occur when transgenic azuki is grown in areas where its wild and weedy relatives occur. The approach used here could be applied to biosafety monitoring of transgenic azuki bean.