Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has become a fundamental procedure for gastrointestinal and lung cancer staging. However, there is growing evidence that micrometastases are present in lymph nodes, which cannot be detected with standard pathological methods. The aim of this study was to evaluate whether hypermethylation gene promoter analysis was feasible on samples obtained by EUS-FNA from lymph nodes, as well as to establish the usefulness of this strategy for the detection of micrometastases in patients with gastrointestinal and non-small cell lung cancer. Suspicious lymph nodes based on EUS findings from consecutive patients with esophageal, gastric, rectal, and non-small cell lung cancer were sampled by EUS-FNA. Hypermethylation analysis of the MGMT, p16(INK4a), and p14(ARF) gene promoter CpG islands were performed by methylation-specific PCR. Effectiveness of conventional cytology, methylation analysis, and their combination were established with respect to the definitive diagnosis. Twenty-seven patients were included, thus representing a total of 42 lymph nodes (esophageal cancer, n = 11; rectal cancer, n = 7; gastric cancer, n = 3; and lung cancer, n = 21). According to definitive diagnosis, 21 (50%) corresponded to metastatic lymph nodes. Sensitivity, specificity, and overall accuracy of conventional cytology were 76%, 100%, and 88%, respectively, whereas the corresponding values for the methylation analysis were 81%, 67%, and 74%, respectively. Combination of both techniques increased sensitivity (90%) but decreased specificity (67%) with respect to conventional cytology. In conclusion, it is feasible to detect occult neoplastic cells in EUS-FNA samples by hypermethylation gene promoter analysis. Moreover, addition of methylation analysis to conventional cytology may increase its sensitivity at the expenses of a decrease in its specificity.