The prevailing cause of α-thalassemia in Southeast Asia is the presence of 3 deletion mutations in the α-globin genes (–SEA, –α3.7 and –α4.2). Current detection methods include gap polymerase chain reaction (PCR), multiplex PCR and real-time PCR with SYBR Green 1 combined with dissociation curve analysis. To improve and simplify a previously published method that requires 4 separate reactions, a duplex PCR assay was designed to detect both the nondeletional and the –SEA alleles. This duplex PCR can successfully identify the nondeletional allele and both the –SEA carrier and homozygous genotypes. The combination of the duplex PCR and 2 gap PCRs (for detection of –α3.7 and –α4.2) can diagnose all types of deletional α-thalassemia. Our method was validated by analysis of 195 DNA samples, the results of which were consistent with prior diagnoses. The developed assay can reliably diagnose α0-thalassemia and all types of deletional α-thalassemia. The diagnostic method is simple, rapid, accurate, automated, inexpensive and has a high throughput.