Williams-Beuren syndrome (WBS) is caused by a 1-2 Mb microdeletion in the region 7q11.23. The clinical presentation may vary and most of the connective tissue abnormalities can be explained by the haploinsufficiency of the ELN gene in this region. The purpose of this study was to determine the value of a polymerase chain reaction assay that uses three polymorphic markers to detect the microdeletion and compare the clinical features. Thirty-two patients with WBS were ascertained accordingly to clinical diagnostic criteria. The markers D7S1870, ELN 17/exon 18, and Hei 1.3/1.4 were designed to detect the heterozygosity in the region 7q11.23. The three markers were informative in 78% and uninformative in 22% of the cases. The most informative marker (69%) was D7S1870, followed by Hei (55%) and ELN 17/exon 18 (44%). The microdeletion was present in 56% and absent in 22% of patients. The craniofacial and cardiovascular abnormalities did not have significant statistical differences in the cases with and without microdeletion. Two of the syndrome characteristics (an overfriendly personality and hyperacusis) were more frequent in the microdeletion group and these differences were statistically significant (p = 0.006 and p = 0.02, respectively). Polymorphic markers might be a good alternative for the molecular diagnosis of WBS in centers where fluorescence in situ hybridization analysis is not available.