The present disclosure relates to calibration assemblies and methods for use with an imaging system, such as an endoscopic imaging system. A calibration assembly includes: an interface for constraining engagement with an endoscopic imaging system; a target coupled with the interface so as to be within the field of view of the imaging system, the target including multiple of markers having calibration features that include identification features; and a processor configured to identify from first and second images obtained at first and second relative spatial arrangements between the imaging system and the target, respectively, at least some of the markers from the identification features, and using the identified markers and calibration feature positions within the images to generate calibration data.