Thalassemias are common inherited blood disorders that have been extensively studied in Asia. Thus far, data on mutations of the HBB gene in Vietnamese patients with β-thalassemia are limited to small studies.We recruited 696 β-thalassemia patients and carriers in southern Vietnam and analyzed for the HBB gene mutations using Sanger sequencing technology.We documented 27 types of known mutations and 10 types of novel variants on 737 alleles out of 1392 surveyed alleles. The three most common mutations, which account for more than ¾ of all mutant alleles, were c.79G A (HbE), c.124_127delTTCT, and c.52A T. The novel variants were mainly located in 5' untranslated region (c.-92delC and c.-67A G) and 3' untranslated region (c.*4C T, c.*116_*117insA, c.*142 T C, c.*156G C, c.*176_*177insA, and c.*247 T C), except for one in intron 2 (c.316-99 T G) and one in exon 3 (c.385delG).We provide here a comprehensive mutation spectrum of the HBB gene in Southern Vietnam, which is crucial for carrier screening and prenatal diagnosis in the future.