Background: Thalassemia, a common autosomal hereditary blood disorder worldwide, mainly contains alpha- and beta-thalassemia. The alpha-globin gene triplicates allele is harmless for carriers, but aggravates the phenotype of beta-thalassemia. Therefore, it is particularly crucial to accurately detect the structural variants of alpha-globin gene clusters.Case report: We reported a 28-year-old man, the proband, with microcytic hypochromic anemia. From pedigree analysis, his mother and sister had hypochromic microcytosis, and his father was normal. Genetic testing of thalassemia identified a novel alpha-globin gene triplicate named alpha alpha alpha(anti4.2del726bp) (NC_000016.10:g.170769_174300dupinsAAAAAA) by third-generation sequencing (TGS) in the proband and his father, which was further validated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. The genotypes of the proband's mother and sister were both -alpha(3.7)/alpha alpha compounded with heterozygous HBB:c.126_129delCTTT. They were categorized as silent alpha-thalassemia with co-inheritance of beta-thalassemia trait. The proband's genotype additionally had the alpha-globin gene triplicates compared with his mother and sister, which increased the imbalance between alpha/beta-globin, so the proband had more severe hematological parameters. The proband's wife was diagnosed as HBA2:c.427T > C heterozygosis, and his daughter had the novel alpha-globin gene triplicates compounded with HBA2:c.427T > C, therefore the girl might be asymptomatic.Conclusion: The identification of the novel alpha-globin gene triplicates provides more insight for the research of thalassemia variants and indicates that TGS has signi?cant advantages on genetic testing of thalassemia for the reliability, accuracy and comprehensiveness.