Abstract β-thalassemia, one of the most common inherited disorders of hemoglobin synthesis in the world, is genetically heterogeneous with over 200 different β-globin mutations worldwide. In this study, we describe a novel frameshift β-thalassemia mutation at codon (cd) 53 (−T) in exon 2 of the β-globin gene in a Chinese Miao family. In this family, all seven heterozygotes with this mutation presented with moderate anemia, jaundice, splenomegaly and elevated hemoglobin A2 levels. None of them had been transfused or carried any other known α/β-globin mutation. Pedigree analysis indicated an autosomal dominant inheritance pattern in this family. Two new haplotypes “−−−−+−+” and “−−+++−+” were identified by restriction fragment length polymorphism (RFLP) haplotype analysis. The former was associated with the cd53 (−T) mutation and the latter only existed in one family member. Thus, a novel frameshift cd53 (−T) mutation may lead to mild thalassemia intermedia even though there is no statistically significant difference in β-globin messenger RNA (mRNA) level between six heterozygotes and six normal subjects.