BACKGROUND: Traditional histological methods for identifying ectopic thymus (ET) have significant limitations including high risk of false negatives. This study aims to evaluate the effectiveness of flow cytometry in detecting ETs in patients undergoing total thymectomy. METHODS: We analyzed 864 samples from 103 patients using flow cytometry and hematoxylin and eosin (H&E) staining. ETs were identified by the presence of CD3 RESULTS: In the discovery set, flow cytometry detected ETs in 69.2% of samples, compared to 23.6% by histological methods. The validation set showed a higher incidence of ETs in myasthenia gravis (MG) patients than in non-MG patients (73.5% vs. 58.0%, CONCLUSIONS: Flow cytometry is a viable alternative for ET detection, providing a novel distribution map that enhances surgical decision-making in MG treatment.