Virtual endoscopy (VE) is a recently developed technique to provide a realistic surface rendering of various organs, which can be applied to the use of three-dimensional (3D) studies of several lesions. However, its advantages in otological disease have not been well investigated. In this study, we evaluated the application of VE in patients with ossicular chain anomalies. Virtual middle ear endoscopy was a time-saving method, however, we needed the appropriate technical procedures of algorithm and reconstruction spacing to generate accurate 3D images of ossicles. We obtained virtual surgical views of middle ear structures and related anomalies, and confirmed by intraoperative findings that these images were mostly compatible with the actual lesions of ossicles. VE allowed an identification of the anatomy of the ossicles and adjacent structures simultaneously. The elements of the stapedial crura were clearly visualized with VE images in 93.3% of normal ears. Pathological ossicular chain findings such as malleus or incus fixation, dislocation and disruption, except footplate fixation were investigated successfully. One possible procedure, using alterable CT value in the obtained VE images on the monitor, is proposed for further detection of fine lesions of the ossicles. These observations suggest that virtual middle ear simulations accurately represent major intraoperative findings. This technique may have an important role in preoperative planning, surgical training, and/or postoperative evaluation in otology.