Deletion mutations of 3.7 kb and 4.2 kb of α-globin gene are the most common causes of α-thalassemia (-α 3.7/, -α 4.2/). A simple, rapid assay by using a single-tube PCR to detect the two deletions has been needed. In this study, a pair of shared primers was designed for α2 and α1 gene but with length-different amplicons (159 bp and 409 bp). On the dissociation curve analysis profile after PCR, there shows two obvious peaks which represent the two different amplicons. Relative copy number of α2 and α1 gene can be deduced from the ratio of the two peaks. A comprehensive diagnosis for α-thalassemia 10 genotypes of deletions can be achieved when combined with a single-tube duplex PCR for detecting −−SEA and non-deletional alleles of αα or α Tα. Besides, a single-tube multiplex PCR, which is a cost-effective version of dual-priming-oligonucleotide based system, was designed for two common mutations of α-thalassemia in China (Hb Constant Spring and Hb Quong Sze), and these two mutations can be identified in samples by use of dissociation curve analysis. In all, using above three PCRs followed by dissociation curve analysis, three deletions and two mutations of α-thalassemia in the populations of southern China and Southeast Asia can be detected for molecular diagnosis or prenatal diagnosis. A blinded study of 163 samples was performed using this new assay and it was concordant with the original methods. This comprehensive molecular assay is simple, rapid, automatic and cost-effective, and can be used to diagnose α-thalassemia in this geographical area.