SS2-2, a hypernodulating soybean mutant, was isolated by EMS mutagenesis from Sinpaldalkong 2. This autoregulation mutant showed greater number of nodules and smaller plant size than its wild type Sinpaldalkong 2. SSR markers were used to identify DNA variation at SSR loci from different soybean LG. The only SSR marker that detected a length polymorphism between SS2-2 and its wild type ancestor was Satt294 on LG C1 instead of LG H, locating a hypernodulating gene. Sequencing data of flanking Satt294 indicated that the size variation was due to extra stretch of TTA repeats of the SSR motif in SS2-2, along with A→G transversion. In spite of phenotypic differences between the wild type and its hypernodulating mutants, genomic DNA polymorphisms at microsatellite loci could not control regulation of nodule formation. The cDNA-AFLP method was applied to compare differential display of cDNA between Sinpaldalkong 2 and SS2-2. After isolation and sequence comparison with many AFLP fragments, several interesting genes were identified. Northern blot analysis, immunolocalization and/or the yeast two-hybrid system with these genes might provide information on regulation of nodule development in SS2-2.