Abstract Purpose To assess the feasibility of thoracoscopic transdiaphragmatic approach for biopsy of all lung lobes and to determine the optimal intercostal space (ICS) for biopsy of each lung lobe. Methods Ten rabbits were positioned in dorsal recumbency. Total thoracoscopy lung biopsy was made combined transdiaphragmatic approach and right ICS approaches. A camera port was made in the transdiaphragmatic approach and the instrument port was made of ICS 7 and ICS 9. A pre tied loop ligature was placed to performed a caudal lung lobe biopsy and to simulate biopsies of the others lung lobes. Results Biopsy of the cranial aspect of the right caudal lung lobe was performed at ICS 9. Simulated biopsy of the accessory lung lobe was performed at ICS 9. Simulated lung biopsy of the right cranial and middle lung lobes was performed at ICS 7. The caudal and dorsal aspect of the right caudal lung lobe was not visualized by telescope at transdiaphragmatic approach, and biopsy was not performed. Conclusions Thoracoscopic transdiaphragmatic approach for lung lobes biopsies was a feasible technique, except for the caudal aspect of the right caudal lung lobe. An ideal intercostal port for biopsy of each right lung lobe was determined.