En-bloc tumor resection is the standard treatment for locally advanced colorectal cancer (CRC). An extensive histopathological assessment is necessary to evaluate the metastatic spread and adjuvant therapy. Sentinel lymph node biopsy decreases the histopathological burden when only sentinel lymph nodes (SLNs) are examined. This study aims to evaluate the spread of a magnetic tracer throughout the lymphatic system after ex vivo injection in en-bloc resected specimens of patients with CRC. To achieve this, lymph nodes (LNs) were quantified using a new magnetic detection method. Fifteen patients with CRC diagnosed with clinically negative LNs were included in this study and received 2–4 ex vivo magnetic tracer injections (total volume of 2mL). Magnetic sample series were acquired to create a look-up table for magnetic tracer quantification. In 80% of the patients, at least one magnetic LN was detected. A total of 33 LNs were marked as magnetic, containing an average of 8.1μμg iron. In 71% of the patients, metastases were found in nonmagnetic LNs. Ex vivo injection leads to sub-optimal tracer spread and therefore inaccurate diagnosis. This study presents a novel magnetic detection method to quantify magnetic tracer in lymph nodes. Detecting the SLNs in en-bloc resected specimens and involving only these LNs in histopathological investigation enable a decrease in healthcare costs or an increased diagnostic potential.