Background: Differences between virtual bronchoscopic navigation (VBN) systems and their impacts on the diagnostic yield of transbronchial biopsy (TBB) of peripheral pulmonary nodules (PPNs) remain unclear. Objectives: To compare the Synapse 3D system (Version 4.4, Fujifilm, Japan) and DirectPath system (Version 2.0, Olympus, Japan) in the VBN application of PPNs. Design: Retrospective study with self-paired design and exploratory study with retrospective cohort design. Methods: The study analyzed patients with PPNs using the Synapse 3D system (Group S) and DirectPath system (Group D) and compared differences between the two groups in bronchial tree reconstruction, navigation pathway planning, and VBN-assisted TBB of PPNs. Results: In all, 289 patients were analyzed ultimately. Bronchial tree reconstruction quality was better in Group S ( p < 0.001). Navigation pathway planning duration in Group S was longer than that in Group D (median 1.35 vs 1.04 s, p < 0.001). Automated navigation pathway planning success rate in Group S was higher than that in Group D (36.7% vs 19.7%, p < 0.001), and CT image reconstruction parameter and nodule diameter, bronchus sign, and distance from the hilum had significant effects on it in both groups. Fifty-six patients in Group S and forty-two patients in Group D were analyzed ultimately. The localization success rate and diagnostic yield of PPNs between the two groups were not significantly different (85.3% vs 91.2% and 67.6% vs 61.8%, respectively, p > 0.05). Conclusion: Synapse 3D system (Version 4.4) and DirectPath system (Version 2.0) had their own merits. Localization success rate and diagnostic yield of VBN-assisted TBB were of no statistical difference for these two VBN systems. Improvements in segmentation algorithms of VBN systems and using the most suitable chest CT scan data for them may be the breakthrough to improve the efficiency of VBN, especially for poor experienced interventional physicians.