OBJECTIVES: Nanopore sequencing shows advantages in detecting single nucleotide variations (SNVs), deletions, and complex structural variants as a single test in thalassemia. However, the performance evaluation or verification of this method remains unestablished, which is essential before clinical utility and panel registration. Here, we developed a classification method for thalassemia mutations, enabling automated interpretation, visual representation, and identification of diverse mutation types. METHODS: We used a total of 36 samples, comprising 32 reference materials and four clinical samples to assess the performance of nanopore sequencing in identifying variants in terms of concordance, precision, and the lower limits of detection. RESULTS: Our analysis successfully identified 19 SNVs, six deletions, and two triplications using nanopore sequencing across all samples. Notably, these variants showed complete concordance of 100% with the genotypes of the reference materials and known results. The precision of nanopore sequencing for detecting thalassemia variants was consistently high, with neither false positive nor false negative observed. Furthermore, the lower limits of detection achieved in our study were 3 ng/μL. CONCLUSIONS: Overall, our study proved that the reference materials can be used to evaluate the performance of nanopore sequencing in identifying thalassemia mutations, and it is necessary to incorporate triplications when utilizing reference materials for performance evaluation of long-read sequencing. The consistent and robust performance of nanopore sequencing in this study demonstrates its potential as a reliable method for comprehensive variant detection in thalassemia and other genetic diseases diagnosis.