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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 0 -thalassemias 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% (239/1046) and 13.34% (279/2092), respectively. Of these, -- THAI and -- CR were relatively rare in this population and comparable to each other with the allelic frequency of 0.14% (3/2092)., Conclusion: This study successfully established a reliable molecular diagnostic platform for genotyping of -- CR , -- SEA , and -- THAI in a single reaction. Additionally, we demonstrated the frequency of -- CR in Thailand for the first time and provided knowledge basis for the planning of severe α-thalassemia prevention and control programs in Thailand, where thalassemia is endemic., (© 2023 John Wiley & Sons Ltd/University College London.)