Although the population prevalence and mutation spectrum of β-thalassemia in most areas of south China have been characterized, the mutations have not been elucidated in Guizhou Province. The aim of this study was to investigate the spectrum of β-thalassemia in this province.We detected and analyzed β-globin gene mutations in 407 β-thalassemia patients and carriers by PCR-based reverse dot blot (RBD) and direct sequencing methods.Twelve types of β-globin gene mutations were detected. Among the 12 different mutations, six mutations are common, accounting for 97% of mutated alleles. The most prevalent mutation is codon 17 (A→T) with an allele frequency of 40.7%. In addition, codon 121 (GAA>TAA), a rare dominant mutation, was detected in a patient with β-thalassemia intermedia for the first time in China.The results of this study will be useful in genetic counseling and prenatal diagnostic service of β-thalassemia in Guizhou Province.