This study has identified and characterized the structure of locus D-A, a noncoding short tandem repeat (STR) region, also known as locus 1-2, in Iranian Entamoeba dispar isolates. This polymorphic locus has been shown to be potentially useful in investigating the molecular epidemiology of Entamoeba histolytica and E. dispar. The genetic polymorphisms in locus D-A in 28 isolates of E. dispar from three different geographic regions of Iran were distinguished using PCR and sequencing, and the results were compared with the E. dispar gene sequences available in GenBank. In all microscopy-positive E. histolytica/E. dispar samples, PCR with species-specific primers was used to amplify a 477-531 bp product, identifying the samples that had E. dispar. Analysis of the sequences revealed a remarkable degree of genetic diversity with regard to size, number and organization of the repeat units among the E. dispar isolates. The sequenced products showed 12 novel E. dispar genotypes, which have been submitted to the GenBank/EMBL/DDBJ database under accession numbers AB354125-AB354136.