Objective: To evaluate the sensitivity and specificity of hemoglobin (Hb) electrophoresis in differentially diagnosing anemia in pregnant athletes, focusing on thalassemia conditions. Methods: This study included 120 participants divided into three groups: Group 1 consisted of 40 pregnant athletes diagnosed with β-thalassemia, Group 2 included 40 pregnant athletes with α-thalassemia, and the control group comprised 40 healthy pregnant athletes, all undergoing routine prenatal examinations from March 2021 to April 2023. Hemoglobin electrophoresis was performed to assess levels of hemoglobin A2 (HbA2) and hemoglobin F (HbF). We compared the expression levels of HbA2 and HbF across the groups, analyzed the positivity rates, and evaluated the diagnostic efficiency using receiver operating characteristic (ROC) curves. Results: Both HbA2 and HbF levels were significantly higher in Group 1 compared to Group 2 and the control group (P < 0.05). The positivity rates for HbA2 and HbF in Group 1 were 30.00% and 25.00%, respectively, which were significantly higher than those in Group 2 (10.00%, 0%) and the control group (8.00%, 7.50%), P < 0.05. The area under the curve (AUC) for differential diagnosis between β-thalassemia and α-thalassemia using combined HbA2 and HbF detection was 0.896, with a 95% confidence interval of 0.834-0.952. The combined detection approach yielded higher sensitivity (94.69%) and specificity (90.12%) than single marker detection (72.26% sensitivity, 68.85% specificity for HbA2 and 85.08% sensitivity, 70.14% specificity for HbF), P < 0.05. Conclusion: The study highlights the efficacy of using Hb electrophoresis in the differential diagnosis of β-thalassemia and α-thalassemia among pregnant athletes. The combined analysis of HbA2 and HbF significantly enhances diagnostic accuracy, providing a valuable tool for managing thalassemia in this specific population.