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Introduction: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked enzymopathy, highly prevalent in the areas where malaria is or has been endemic. Prevalence of G6PD deficiency and characterization of G6PD variants in females from previously malaria-endemic areas of northeastern Thailand remain unstudied., Methods: Prevalence of G6PD deficiency was determined by a fluorescent spot test (FST), quantitative G6PD activity assay, and multiplex allele-specific (AS)- and restriction fragment length polymorphic (RFLP)-PCR developed for detection of common G6PD variants in the Thai population., Results: Prevalence of G6PD deficiency in female samples (n = 355) was 18% by FST, 29.6% by quantitation of G6PD activity, and 28.1% by PCR-based genotyping. The most common variant was G6PD Viangchan (54%), followed by G6PD Canton (11%) and G6PD Union (11%); in addition, a novel heterozygous variant, G6PD Khon Kaen (c.305T>C, p.F102S), was identified. The majority of heterozygotes expressed G6PD activity within the intermediate deficiency range (30-70% median of normal enzyme activity)., Conclusion: High prevalence of G6PD deficiency was present in females from northeastern Thailand, the majority being due to heterozygosity of G6PD variants. The findings will have a bearing on the inclusion of primaquine in antimalarial-based policies for malaria elimination in populations with a high prevalence of G6PD deficiency., Competing Interests: Competing interests: The authors declare no conflict of Interest.