The purpose of this study is evaluating registration accuracy of evaluation environment of Digital and Analog Pathology (eeDAP). eeDAP was developed to help conduct studies in which pathologists view and evaluate the same fields of view (FOVs), cells, or features in a glass slide on a microscope and in a whole slide image (WSI) on a digital display by registering the two domains. Registration happens at the beginning of a study (global registration) and during a study (localregistration). The global registration is interactive and defines the correspondence between the WSI and stage coordinates. The local registration ensures the pathologist evaluates the correct FOVs, cells, and features. All registrations are based on image-based normalized cross correlation.This study evaluates the registration accuracy achieved throughout a study. To measure the registration accuracy, we used an eyepiece ruler reticle to measure the shift distance between the center of the eyepiece and a target feature expected in the center. Two readers independently registered 60 FOVs from 6 glass slides, which covered different tissue types, stains, and magnifications. The results show thatwhen the camera image is in focus, the registration was within 5micrometers in more than 95% of the FOVs. The tissue type, stain, magnification, or readerdid not appear to impact local registration accuracy. The registration error was mainly dependent on the microscope being in focus, the scan quality, and the FOVcontent (unique high-contrast structures are better than content that is homogeneous or low contrast).