Diffuse optical imaging using non-ionizing radiation is a non-invasive method that shows promise towards breast cancer diagnosis. Hand-held optical imagers show potential for clinical translation of the technology, yet they have not been used towards 3D tomography. Herein, 3D tomography of human breast tissue in vivo is demonstrated for the first time using a hand-held optical imager with automated coregistration facilities. Simulation studies are performed on breast geometries to demonstrate the feasibility of 3D tomographic imaging using a hand-held imager under perfect (1:0) and imperfect (100:1, 50:1) fluorescence absorption contrast ratios. Experimental studies are performed in vivo using a 1 µM ICG filled phantom target placed non-invasively underneath the flap of the breast tissue. Results show the ability to perform automated tracking and coregistered imaging of human breast tissue (with tracking accuracy on the order of ∼1 cm). Three-dimensional tomography results demonstrated the ability to recover a single target placed at a depth of 2.5 cm, from both the simulated (at 1:0, 100:1 and 50:1 contrasts) and experimental cases on actual breast tissues. Ongoing efforts to improve target depth recovery are carried out via implementation of transmittance imaging in the hand-held imager.