SummaryFluorescence‐based DNA sequence analysis was developed for identification of β‐globin gene mutations in the Thai population. The β‐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 β‐thalassemia disease, β‐thalassemia/hemoglobin (Hb) E and Hb variants. Twenty‐five different β‐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 β‐globin gene mutations, which is essential for prevention and control of thalassemia and hemoglobinopathy in Thailand.