Introduction: There remains no standard imaging method that allows computer-assisted surgery of the cochlea in real time. However, recent evidence suggests that high-frequency ultrasound (HFUS) could permit real-time visualization of cochlear architecture. Registration with an imaging modality that suffers neither attenuation nor conical deformation could reveal useful anatomical landmarks to surgeons. Our study aimed to address the feasibility of an automated three-dimensional (3D) HFUS/microCT registration, and to evaluate the identification of cochlear structures using 2D/3D HFUS and microCT. Methods: MicroCT, and 2D/3D 40 MHz US in B-mode were performed on ex vivo guinea pig cochlea. An automatic rigid registration algorithm was applied to segmented 3D images. This automatic registration was then compared to a reference method using manual annotated landmarks placed by two senior otologists. Inter- and intrarater reliabilities were evaluated using intraclass correlation coefficient (ICC) and the mean registration error was calculated. Results: 3D HFUS/microCT automatic registration was successful. Excellent levels of concordance were achieved with regards intra-rater reliability for both raters with micro-CT and US images (ICC ranging from 0.98 to 1, p < 0.001) and with regards inter-rater reliability (ICC ranging from 0.99 to 1, p < 0.001). The mean HFUS/microCT automated RE for both observers was 0.17 ± 0.03 mm [0.10-0.25]. Identification of the basilar membrane, modiolus, scala tympani, and scala vestibuli was possible with 2D/3D HFUS and micro-CT. Conclusions: HFUS/microCT image registration is feasible. 2D/3D HFUS and microCT allow the visualization of cochlear structures. Many potential clinical applications are conceivable.