Diffuse optical tomography (DOT) has been studied for use in the detection of breast cancer, cerebral oxygenation, and cognitive brain signals. As optical imaging studies have increased significantly, acquiring imaging data in real time has become increasingly important. We have developed frequency-division multiplexing (FDM) DOT systems to analyze their performance with respect to acquisition time and imaging quality, in comparison with the conventional time-division multiplexing (TDM) DOT. A large tomographic area of a cylindrical phantom 60 mm in diameter could be successfully reconstructed using both TDM DOT and FDM DOT systems. In our experiment with 6 source-detector (S-D) pairs, the TDM DOT and FDM DOT systems required 6.18 and 1 s, respectively, to obtain a single tomographic data set. While the absorption coefficient of the reconstruction image was underestimated in the case of the FDM DOT, we experimentally confirmed that the abnormal region can be clearly distinguished from the background phantom using both methods.