Approximately 20% - 30% of histological lymph node-negative patients with colorectal cancer relapse at five years aftersurgical treatment. This recurrence is likely due to occult nodal disease undetected by standard histopathological practice which hasimplications in terms of the clinical management of patients. Lymph nodes were collected from colectomy specimens. A central section from each lymph node washistologically examined following haematoxylin-eosin staining and the remaining tissue was subjected to OSNA - one step nucleic acidamplification analysis. A total of 1046 lymph nodes from 59 pN0 patients were assessed. Of these, 753 lymph nodes were examined by bothmethods. The median number of lymph nodes assessed with OSNA - one step nucleic acid amplification was 12 (IQR: 7;16). AmongpN0 patients, 17 had OSNA - one step nucleic acid amplification-positive lymph nodes, resulting in a positive molecular staging rateof 28.8% (95% CI: 17.8 - 42.1). Among these patients, 12 (70.59%) were molecular-staged as pN1 and 5 (29.41%) were molecularstaged as pN2. The tumour burden of lymph nodes assessed with OSNA - one step nucleic acid amplification ranged from 270 to17 000 cytokeratin 19 mRNA copies/μL. Most of these patients (88.2%) were found to have lymph nodes with micrometastases only(250 - 4999 copies/μL). We provide the results from the first study of the use of the OSNA - one step nucleic acid amplification assay in colorectalcancer patients in Portugal. Our results are in-line with other international studies, showing the improvement on patients' staging bymolecular examination of lymph nodes. In our study, 28.8% of patients with histologically negative lymph nodes were found to have metastatic lymph nodes usingOSNA - one step nucleic acid molecular assessment. OSNA - one step nucleic acid assay allows a more accurate staging of patientswith colorectal cancer and standardizes lymph node assessment.