Beta-thalassemia is the most predominant genetic defect in Greece. In this study, we investigated the heterogeneity and the frequency of beta-thalassemia mutations among 3796 heterozygotes detected in the course of DNA based diagnoses. The diagnostic strategy included Denaturing Gradient Gel Electrophoresis (DGGE), Allele Specific Oligonucleotide Hybridization (ASO), GAP PCR, Restriction Enzyme (RE) analysis and direct sequencing and led to 100% identification of the underlying molecular lesion. Six out of 33 different beta-globin defects identified accounted for more than 91.4% of the total beta-thalassemia chromosomes in Greece. The beta-globin gene mutations cd29 C-->T, IVS-I-2 T-->C, IVS-I-5 G-->T, cd37 G-->A and poly A Kurdish AATAAA-->AATAAG are for the first time reported in Greece, whereas cd7 GAG-->TAG is a new beta(0)-thalassemia mutation detected in an adult man from Albania residing in Greece. Three DNA single nucleotide polymorphisms (IVS-I-85 T-->C, IVS-I-91 C-->T and IVS-I-108 T-->C) were also revealed; among these, IVS-I-85 T-->C and IVS-I-91 C-->T are new and described for the first time worldwide.