The invention provides for surface mapping of in-vivo imaging subjects using a single camera and an illuminator that projects a plurality of targets such as spots on the subject. By limiting the depth-of-field of the camera lens, or of the illuminator optics, or both, a spatial plane is defined in which the spots are most sharply in focus. Controlled displacement of this plane relative to the subject is achieved through movement of the mechanical stage on which a subject is placed; or through movement of the best-focus plane by adjustment of the camera, lens, or illuminator optics. Images are taken at several relative positions of the best-focus plane and the subject, and the height of individual points on the subject is determined through analysis of focus, given the known displacements. A mesh or other surface can be constructed from individual point locations, to provide a surface map of the subject. Accuracy of 0.5 mm can be readily attained for mice and similarly sized subjects.