This study reports the molecular characterization of thalassemia syndromes in Serbian and Montenegrin populations. We identified eight β-thalassemia mutations [codon 39 (C→T), IVS-I-110 (G→A), IVS-II-745 (C→G), codon 44 (-C), –87 (C→G), IVS-II-1 (G→A), IVS-I-6 (T→C), IVS I-1 (G→A)] in 70 members of 29 families using polymerase chain reaction, reverse dot blot, amplification refractory mutation system and direct sequencing analysis. Hemoglobin (Hb) Lepore was found to be the most common cause of the thalassemia phenotype. Hb Sabine and α-thalassemia were detected as well. We also studied β-globin gene cluster haplotypes and their association with the most common mutations. A novel haplotype associated with the Hb Lepore gene was identified. The results presented herein allowed the implementation of a prenatal diagnosis program in Serbia and Montenegro.