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This is particularly important in regions where the prevalence of G6PD deficiency exceeds 3%\u20135%, including Southeast Asia and Thailand. While quantitative phenotypic tests can identify women with intermediate activity who may be at risk, they cannot unambiguously identify heterozygous females who require appropriate counselling. This study aimed to develop a genetic test for G6PD deficiency using high\u2010resolution melting curve analysis, which enables zygosity identification of 15 <jats:italic>G6PD<\/jats:italic> alleles. In 557 samples collected from four locations in Thailand, the prevalence of G6PD deficiency based on indirect enzyme assay was 6.10%, with 8.08% exhibiting intermediate deficiency. The developed high\u2010resolution melting assays demonstrated excellent performance, achieving 100% sensitivity and specificity in detecting <jats:italic>G6PD<\/jats:italic> alleles compared with Sanger sequencing. Genotypic variations were observed across four geographic locations, with the combination of c.1311C&gt;T and c.1365\u201013T&gt;C being the most common genotype. Compound mutations, notably <jats:italic>G6PD<\/jats:italic> Viangchan (c.871G&gt;A, c.1311C&gt;T and c.1365\u201013T&gt;C), accounted for 15.26% of detected mutations. The high\u2010resolution melting assays also identified the double mutation <jats:italic>G6PD<\/jats:italic> Chinese\u20104\u2009+\u2009Canton and <jats:italic>G6PD<\/jats:italic> Radlowo, a variant found for the first time in Thailand. Biochemical and structural characterisation revealed that these variants significantly reduced catalytic activity by destabilising protein structure, particularly in the case of the Radlowo mutation. The refinement of these high\u2010resolution melting assays presents a highly accurate and high\u2010throughput platform that can improve patient care by enabling precise diagnosis, supporting genetic counselling and guiding public health efforts to manage G6PD deficiency\u2014especially crucial in malaria\u2010endemic regions where 8\u2010aminoquinoline therapies pose a risk to deficient individuals.<\/jats:p>","DOI":"10.1111\/tmi.14105","type":"journal-article","created":{"date-parts":[[2025,3,13]],"date-time":"2025-03-13T07:09:21Z","timestamp":1741849761000},"page":"437-457","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Improved genetic screening with zygosity detection through multiplex high\u2010resolution melting curve analysis and biochemical characterisation for <scp>G6PD<\/scp> 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