High resolution, 100 frames/sec intravascular optical coherence tomography (IVOCT) can distinguish plaque types, but further validation is needed, especially for automated plaque characterization. We developed experimental and 3D registration methods, to provide validation of IVOCT pullback volumes using microscopic, brightfield and fluorescent cryoimage volumes, with optional, exactly registered cryo-histology. The innovation was a method to match an IVOCT pullback images, acquired in the catheter reference frame, to a true 3D cryo-image volume. Briefly, an 11-parameter, polynomial virtual catheter was initialized within the cryo-image volume, and perpendicular images were extracted, mimicking IVOCT image acquisition. Virtual catheter parameters were optimized to maximize cryo and IVOCT lumen overlap. Local minima were possible, but when we started within reasonable ranges, every one of 24 digital phantom cases converged to a good solution with a registration error of only +1.34±2.65μm (signed distance). Registration was applied to 10 ex-vivo cadaver coronary arteries (LADs), resulting in 10 registered cryo and IVOCT volumes yielding a total of 421 registered 2D-image pairs. Image overlays demonstrated high continuity between vascular and plaque features. Bland- Altman analysis comparing cryo and IVOCT lumen area, showed mean and standard deviation of differences as 0.01±0.43 mm2. DICE coefficients were 0.91±0.04. Finally, visual assessment on 20 representative cases with easily identifiable features suggested registration accuracy within one frame of IVOCT (±200μm), eliminating significant misinterpretations introduced by 1mm errors in the literature. The method will provide 3D data for training of IVOCT plaque algorithms and can be used for validation of other intravascular imaging modalities.