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Introduction:The α 0 -thalassemia 44.6 kb or Chiang Rai (--CR ) deletion has been reported in northern Thailand and is capable of causing hemoglobin (Hb) H disease and a lethal α-thalassemia genotype, Hb Bart's hydrops fetalis, in this region.However, there are no current data regarding the frequency of --CR nationwide due to a lack of effective diagnostic assay. Therefore, this study aimed to develop a reliable platform for simultaneous genotyping of --CR and two common α 0thalassemias in Thailand (--SEA and --THAI ) and investigate the frequency of --CR across Thailand.Methods: Multiplex gap-PCR assay and five renewable plasmid DNA controls for --CR , --SEA , --THAI , α2-globin (HBA2), and β-actin (ACTB) were newly developed and validated with reference methods. The developed assay was further tested on 1046 unrelated individuals with a reduced mean corpuscular volume (MCV) of less than 75 fl for investigating genotypic and allelic spectrum of --CR . Results: Our developed assay showed 100% concordance with reference methods. The results were valid and reproducible throughout hundreds of reactions.Comparison of the genotypic and allelic spectra revealed that heterozygous --SEA (--SEA /αα) and --SEA alleles were dominant with the frequency of 22.85%