Fluorescence-based DNA sequence analysis was developed for identification of beta-globin gene mutations in the Thai population. The beta-globin gene was directly sequenced in two runs and the sequencing electropherogram allowed unambiguous detection of nucleotide substitutions, frameshifts, and small insertions/deletions in heterozygote and homozygote. The method was validated and successfully applied in routine analysis of 416 individuals with beta-thalassemia disease, beta-thalassemia/hemoglobin (Hb) E and Hb variants. Twenty-five different beta-globin gene mutations were identified. Two Hb variants, Hb Tacoma [codon 30 (G-T)] and Hb Tende [codon 124 (C-T)], were also identified for the first time in a Thai population. Automated fluorescence-based DNA sequence analysis provides a rapid and reliable method for identification of common, rare and unknown beta-globin gene mutations, which is essential for prevention and control of thalassemia and hemoglobinopathy in Thailand.