We previously reported that MUC2 is a useful marker in the detection of lymph node micrometastasis (LMM) in gastric cancer. To improve the detection rate, we focused on a TFF1 gene. We used the duplex reverse transcriptase-polymerase chain reaction (RT-PCR) method with MUC2 and TFF1 genes to detect LMM in histologically node negative (pN0) early gastric cancer (EGC) and evaluated their effectiveness. A total of 310 lymph nodes from 33 patients with pN0 EGC were analyzed. All carcinoma specimens were positive for MUC2 and/or TFF1. The positive rate of TFF1 was significantly higher than MUC2 in the undifferentiated carcinoma specimens (p=0.002). The detection rate of duplex RT-PCR with MUC2 and TFF1 was higher than that of MUC2 or TFF1 alone. In mucosal cancer, 7 cases were positive for duplex RT-PCR. Of these 7 cases, 3 were MUC2-positive/TFF1-negative while 4 were MUC2-negative/TFF1-positive. LMMs were not detected in elevated-type primary mucosal cancers with a 20-30-mm diameter. The duplex RT-PCR assay with both MUC2 and TFF1 provides a higher LMM detection rate than either MUC2 or TFF1 alone, especially in mucosal gastric cancer. LMM detected in this manner may prove useful in broadening the indications for endoscopic mucosal resection in elevated-type cancer.