We present a detailed survey of the diversity of β-globin gene haplotypes in carriers of the most common β-thalassemia mutations (Hb Lepore Boston Washington-33%, codon 39 -22% and IVS-I-110 -17%) and normal betaA/betaA individuals of Serbian descent. We have studied eight linked polymorphic restriction enzyme sites along beta globin gene cluster (Hinc II/ε, Xmn I⁄ 5'Gγ, Hind III/Gγ, Hind III/Aγ, Hinc II/ψβ, Hinc II/3'ψβ, Ava II/β, Bam HI/3'β), as well as intragenic β-globin gene sequence diversity (frameworks) by using PCR-RFLP and sequencing analysis. Four different haplotypes associated with β-thalasse mia mutations were detected; two of them were typical of Mediterranean countries (I and V according to Orkin), and two were novel haplotypes, A (+--+--+--) and B (+--+--++). All the Lepore BW chromosomes were of the identical haplotype -haplotype A. Comparison of this novel haplotype associated with the Hb Lepore BW mutation in Serbia with haplotypes reported to date showed that it differs from all of them. These data support the hypothesis of the multicentric origin of this mutation. Haplotype analysis revealed that the codon 39 thalassemia mutation was associated with haplotype I and framework 1. However, in one family, codon 39 chromosomes were found to be associated with framework 2. Two different haplotypes (I and B) were associated with the IVS-I-110 thalassemic mutation. We presume that two most common Mediterranean mutations, codon 39 and IVS-I-110, have probably been introduced into Serbian population from Italy and Turkey, respectively, through historically documented migrations and settlements.