Background: The Hong Kongαα (HKαα) allele is a complex structural rearrangement of the α-globin gene containing -α 3.7 and ααα anti 4.2 crossover junctions. Clinically, individuals carrying the HKαα allele are often misdiagnosed or missed using conventional thalassemia gene detection technology. This study aims to identify and validate different HKαα thalassemia subtypes using third-generation sequencing (TGS) technology.Methods: Between January 2015 and June 2021, 32 patients suspected of having HKαα thalassemia were included in this study. Genomic DNA was extracted, and gap-polymerase chain reaction (PCR), two-round nested PCR, multiplex ligation-dependent probe amplification (MLPA), and TGS were used for thalassemia gene detection. Results:The results of HKαα/αα and HKαα/-α 3.7 were similar to -α 3.7 /αα using the gap-PCR method. Tworound nested PCR could be used to verify the HKαα gene, but could not distinguish the subtypes of HKαα thalassemia. The MLPA assay was used to detect the change in the copy number of the α-globin gene, but it could not determine whether -α 3.7 and ααα anti 4.2 were in cis or in trans. Long-read TGS technology could accurately detect the HKαα allele and distinguish the genotypes of HKαα/αα, HKαα/-α 3.7 , HKαα/-α 4.2 , and HKαα/--SEA without pedigree analysis. The contiguous sequence of the HKαα allele was detected using the TGS approach. This study also demonstrated that individuals with HKαα/αα and β N /β N genotypes tended to have normal hematological phenotypes.Conclusions: Long-read TGS is a reliable and efficient approach for accurate detection of HKαα thalassemia, which can be widely used in clinical practice. Accurate molecular diagnosis of HKαα thalassemia will benefit clinical genetic counseling and prenatal diagnosis.