Accurate identification of tumor deposits in lymph nodes is important for correct colon cancer staging. Molecular lymph node investigation with one-step nucleic acid amplification (OSNA) could allow more precise lymph node analysis. Promising results from breast cancer demonstrated better detection rates of tumor deposits when using OSNA compared to conventional lymph node staging. However, it was unknown whether this method translates into better survival rates of patients with colon cancer. The here presented study investigated a group of patients with colon cancer from three European centres on a long-term basis, staged with OSNA and Haematoxylin & Eosin (H&E). One-step nucleic acid amplification failed to outperform conventional H&E work-up regarding its prognostic value, contrasting findings in other cancer types. However, future research should focus on the informative value of the total tumor load of the resected lymph nodes and the respective mRNA thresholds in order to detect small but relevant metastatic deposits what may increases the prognostic value of this molecular detection method.