Uniportal video-assisted thoracic surgery (VATS) is gaining popularity among thoracic surgeons, but the limited space in the thorax of children makes uniportal VATS difficult to perform. The purpose of this study was to evaluate procedural and outcome differences between a modified uniportal VATS (MU-VATS) and three-port VATS (TP-VATS) for peripheral lung nodule biopsy in pediatric cancer patients. This is an Institutional Review Board-approved retrospective analysis of all consecutive MU-VATS and TP-VATS peripheral lung nodule biopsies performed at a single institution between June 2014 and December 2016. Patients with diffuse lung disease who underwent a lung biopsy were excluded. Over a 30-month period, 22 patients with a median age of 12 years (range, 7-21) underwent MU-VATS or TP-VATS for excisional biopsy of a peripheral lung nodule. MU-VATS lung biopsy was attempted in 11 patients and TP-VATS lung biopsy in the remaining 11. Both groups were comparable with regard to demographics, primary diagnosis, purpose of biopsy, and lung nodule location. MU-VATS demonstrated no difference when compared with TP-VATS lung biopsy in operative time (54 versus 62 min, P = .899), estimated blood loss (14 versus 15 mL, P = .587), pain score (2.8 versus 2.9, P = .717), and discharge day (1.3 versus 1.2 days, P = .572). No difference existed between groups with regard to conversion, need for intraoperative blood transfusion, and duration of chest tube. Complications including pneumothorax (n = 2) and subcutaneous emphysema (n = 1) were only seen in the TP-VATS group. MU-VATS can be safely utilized for biopsy of peripheral lung nodules in pediatric cancer patients without increasing procedural duration, hospitalization, pain scores, or need for intraoperative blood transfusion. Further studies need to evaluate the theoretical cosmetic advantage from a single surgical scar.