Malignant pleural effusion (MPE) and blood samples can be used as a practical source for detection of epidermal growth factor receptor (EGFR) mutations in patients with advanced non-small cell lung cancer. We compared EGFR mutation status of cell blocks, cell-free fluid of MPE, and plasma from patients with lung adenocarcinoma. We obtained paired samples of MPE and plasma from 14 pathologically-confirmed lung adenocarcinoma patients. Peptide nucleic acid (PNA)-mediated real-time polymerase chain reaction (RT-PCR) clamping was performed for determination of EGFR mutation status. EGFR mutations were detected in five (35.7%) cell blocks of MPE, which showed results identical to those of the corresponding cell-free fluid, whereas mutations were detected in the plasma of only two (40.0%) of the five patients. Of seven patients treated with EGFR tyrosine kinase inhibitors (TKIs), EGFR mutations were detected in cell blocks, cell-free fluid of MPE, and plasma for only one of the four patients who responded to EGFR TKIs, while mutations were detected only in cell blocks of MPE and cell-free fluid of the three remaining patients. Our results suggest that detection of EGFR mutations in cell-free pleural fluid from lung adenocarcinoma patients using highly sensitive methods may be feasible, but that analysis of free plasma may lead to undetected mutations and misdiagnosis.