Objective: To investigate the application of high performance liquid chromatography (HPLC) in the screening of thalassemia in Shanghai. Methods: The hemoglobin of 135 patients with suspected thalassemia were analyzed by using HPLC. Gene detection of thalassemia by GAP-PCR and reverse dot blot (RDB) were simultaneously performed. Results: Using gene detection as a gold standard, when HbA₂>3.7% was taken as the cutoff value for diagnosis of β-thalassemia carriers by HPLC, the sensitivity and specificity were 100%, and 98.41%, and the positive predictive value and negative predictive value were 98.0% and 100%, respectively. If <1.7% was taken as the cutoff value of HbA₂ for diagnosis of α-thalassemia （HbH disease） by HPLC, the indice above-mentioned were 100%, 100%, 100%, 100%, respectively. Out of 135 samples, there were 8 cases being detected a HbH peak. Conclusion: Results of HPLC detection are in excellent agreement with those of the gene detection in the diagnosis of β-thalassemia and HbH disease, while certain false negative rate may occur in the diagnosis of minor α-thalassemia or α-thalassemia trait.