α-thalassemia is an inherited blood disorder that is most frequently found in Southeast Asian populations. In Thailand, molecular characterization can diagnose most patients with α-thalassemia; however, several atypical patients are also observed in routine analyses. Here, we characterized α-thalassemia mutations among 137 Hemoglobin H (Hb H) disease patients and three fetuses of Hb Bart’s hydrops, a fatal clinical phenotype of α-thalassemia. Specifically, we performed multiplex ligation-dependent probe amplification (MLPA) followed by direct DNA sequencing. We noticed common genotypes in 129 patients and eight patients had rare Hb H disease caused by compound heterozygous α0-thalassemia (--CRor --SAdeletion) with α+-thalassemia (-α3.7/-α4.2/αConstant Springα). Furthermore, two affected fetuses had the --SA/--SEAand one had the --CR/--SEAgenotypes. Next, we developed and validated a new multiplex gap-PCR and applied this method to 844 subjects with microcytic red blood cells (RBCs) from various parts of Thailand. The frequency of heterozygous α0-thalassemia was dominated by --SEA363/844 (43%), followed by --THAI3/844 (0.4%), --SA2/844 (0.2%), and --CR2/844 (0.2%) mutations. These findings suggest that aforementioned four mutations should be routinely applied to increase the effectiveness of diagnosis and genetic counseling in this region.