Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand comformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G→A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G→A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T→C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G→A (G6PD Viangchan) was observed mainly in Lao, and 592 C→T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A→G (G6PD Gaohe), 1003 G→A (G6PD Chatham), 1360 C→T (G6PD Union), 1376 G→T (G6PD Canton), and 1388 G→A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.