Objective: Accelerated partial breast irradiation is an emerging treatment option for early stage breast cancer. With accelerated partial breast irradiation, patient setup, and target registration accuracy is vital. The current study compared various methods for isocenter placement accuracy. Methods and Materials: Twenty-three patients treated on an institutional-approved partial breast irradiation protocol were monitored at each treatment fraction. All patients included in this study underwent clip placement at the time of surgery. Patients underwent computed tomographic simulation and surface contours were used to reconstruct a reference surface map. At the treatment machine, patients were initially positioned by laser alignment to tattoos. Orthogonal kilovoltage imaging of the chest wall, followed by video surface mapping of the breast, was performed. This video surface map was matched to the reference surface map to adjust the couch position. Verification orthogonal chest wall imaging and video surface mapping was again performed. The accuracy of setup by laser, orthogonal imaging of the chest wall, and surface alignment was retrospectively compared using the centroid clip position as the reference standard. The impact of setup error by surface alignment and by orthogonal kilovoltage imaging on planning target volume coverage was then calculated. Results: Laser-based positioning resulted in a residual setup error of 3.9 ± 3.7 mm, 4.6 ± 3.9 mm, and 4.3 ± 4.5 mm in the posterior-anterior (P-A), inferior-superior (I-S), and left-right (L-R) directions, respectively, using clips as the reference standard. Setup based on bony anatomy with orthogonal imaging resulted in residual setup error of 3.2 ± 2.9 (P-A), 4.2 ± 3.5 (I-S), and 4.7 ± 5.3 mm (L-R). Setup with video surface mapping resulted in a residual setup error of 1.9 ± 2.2, 1.8 ± 1.9, and 1.8 ± 2.1 mm in the P-A, I-S, and L-R directions, respectively. Vector spatial deviation was 8.8 ± 4.2, 8.3 ± 3.8, and 4.0 ± 2.3 mm with laser, chest wall on board imaging, and video surface mapping based setup, respectively. Setup by video surface mapping resulted in improved dosimetric coverage of the planning target volume when compared with orthogonal imaging of the chest wall (V100 96.0% ± 0.1% vs 89.3% ± 0.2%; V95 99.7% ± 0.01% vs 98.6% ± 0.01%, . P < .05). Conclusions: Video surface mapping of the breast is a more accurate method for isocenter placement in comparison to conventional laser-based alignment or orthogonal kilovoltage imaging of the chest wall.