Down syndrome is one of the most common causes of mental retardation observed in approximately 1/700 live birth. The use of two or more STR markers related to chromosome 21 facilitates the diagnosis of Down syndrome within about six hours from the collection of the samples. This is the first study has been performed in Iranian population to assess the diagnostic value of using small tandem repeat (STR) markers assays for the rapid detection of chromosome 21 trisomy and compare its application to the conventional cytogenetic analysis. Eighty seven cases with Down syndrome and 120 normal controls have been tested by conventional karyotyping and molecular technique using five STR markers (D21S11, D21S1414, D21S1440, D21S1411, D21S1412) located on the long arm of chromosome 21. The heterozygosis of these tested markers has been calculated in tested Iranian population. All tested cases had free chromosome 21 trisomy in cytogenetic analysis from them 3 cases were mosaic for chromosome 21 trisomy. In molecular analysis 85% generated three allelic pattern, 14% dialleilc and 1% monoallelic pattern by using five molecular markers. Diagnosis of chromosome 21 trisomy was achieved for 99% of cases by molecular method, from them 3.5% of cases were mosaic and provided diallelic pattern. We have found a specificity of 100% and sensitivity of 99% for molecular diagnosis of Down syndrome using highly polymorphic STR markers. This technique is cheaper, quicker, no need of alive or sterile samples and useful for wide screening of newborns in nurseries. Molecular method accurately determines trisomy 21 especially when cytogenetic approach is not suitable in the case of fixed or dead tissues, small number of cells such as those taken from early amniocentesis or cells separated from maternal blood for noninvasive prenatal diagnosis. Although molecular diagnosis of Down syndrome targeted specifically to chromosome 21 trisomy but it can exclude the presence of the most frequent chromosomal disorder. The result of this investigation has documented the diagnostic advantages of this approach to perform even for prenatal test or in parallel with cytogenetic analysis.