As a Class II medical device, whole-slide imaging (WSI) systems are emerging to succeed light microscopes used by pathologists in the past decades by digitalizing histological tissue slides into millions of pixels saved in a WSI file. Unlike the standard image file formats such as JPEG or TIFF, a WSI file usually consists of hundreds of compressed images of different magnification levels and focal planes that need to be decompressed, stitched, scaled, and colormanaged to reproduce the view demanded by the user with zooming and panning operations. Currently, most WSI files are stored in proprietary file formats, due to the lack of adopting a standard WSI file format, which hinders the development of third-party WSI viewers by making it difficult to interpret WSI files faithfully. To examine the fidelity of third-party WSI viewers, in this study, three freely available viewers, Sedeen, QuPath, and ASAP, were compared with the factory viewer, NDP, at the pixel level. A software tool was developed to register and calculate the 1976 CIE color difference for each pixel between two viewers. The average color differences were found as 1.30, 18.69, and 18.79 ΔE for Sedeen, QuPath, and ASAP, respectively.