Purpose: To find the optimal slice thickness (Δτ) setting for patient registration with kilovoltage cone-beam CT (kVCBCT) on the Varian On Board Imager (OBI) system by investigating the relationship of slice thickness to automatic registration accuracy and contrast-to-noise ratio. Materials and method: Automatic registration was performed on kVCBCT studies of the head and pelvis of a RANDO anthropomorphic phantom. Images were reconstructed with 1.0 ≤ Δτ (mm) ≤ 5.0 at 1.0 mm increments. The phantoms were offset by a known amount, and the suggested shifts were compared to the known shifts by calculating the residual error. A uniform cylindrical phantom with cylindrical inserts of various known CT numbers was scanned with kVCBCT at 1.0 ≤ Δτ (mm) ≤ 5.0 at increments of 0.5 mm. The contrast-to-noise ratios for the inserts were measured at each Δτ. Results: For the planning CT slice thickness used in this study, there was no significant difference in residual error below a threshold equal to the planning CT slice thickness. For Δτ > 3.0 mm, residual error increased for both the head and pelvis phantom studies. The contrast-to-noise ratio is proportional to slice thickness until Δτ = 2.5 mm. Beyond this point, the contrast-to-noise ratio was not affected by Δτ. Conclusion: Automatic registration accuracy is greatest when 1.0 ≤ Δτ (mm) ≤ 3.0 is used. Contrast-to-noise ratio is optimal for the 2.5 ≤ Δτ (mm) ≤ 5.0 range. Therefore 2.5 ≤ Δτ (mm) ≤ 3.0 is recommended for kVCBCT patient registration where the planning CT is 3.0 mm