The presence of nodal metastasis is an important prognostic factor for malignant melanoma and causes a 50% decrease in survival. Patients found not to have lymph node metastasis as a result of sentinel lymph node biopsy but develop regional lymph node metastasis in the follow-up period are called false negative sentinel. The hypothesis of this study was that excisional biopsy in patients with large lesions may change the lymphatic pathway and therefore cause false negative results. A total of 40 Sprague-Dawley rats were divided into 4 groups: the control group, Group 1 (10 mm excision), Group 2 (15 mm lesion excision), and Group 3 (20 mm excision). Sentinel lymph nodes were found via imaging with nanocolloidal albumin including 99mTc and indocyanine green (ICG) before the excision and at 3 weeks after the excision. Evaluations were performed to determine whether or not they were the same nodes. The false sentinel node detection rate in Group 3 was significantly higher than in both the control group and Group 1 ( < 0.05). No significant difference was detected between Group 2 and 3 according to false sentinel node detection rate. There were differences between preoperative and postoperative detection of sentinel lymph nodes found by radioactive and ICG methods but no significant difference was detected in the Kappa agreement coefficient. It can be suggested that incisional biopsy is performed on large lesions initially, followed by wide excision combined with SLNB at the second stage. This can help to prevent changes in the lymphatic pathway and therefore to decrease false negativity rates caused by the previous surgery.