The size of vesicles and amount of gas they contain is a major factor in the acoustic scattering of Sargassum plants. Here we investigate the morphological character of the vesicles and gas, and their theoretical and experimental target strength (TS) for two Sargassum species, Sargassum macrocarpum and S. siliquastrum. Both species are abundant in the coastal waters of Japan. A total of 593 vesicles were measured using a soft X-ray system, and their gas shapes were applied to two theoretical TS models (non-resonance and resonance). Experimental TS were obtained at 70 and 200 kHz using quantitative echo sounders in a freshwater tank. Gas fields were found in 418 vesicles, and the gas radius and external size of the vesicles were linearly related in both species. The theoretical TS of the non-resonance model was similar to the experimental TS of large vesicles (≥4.1 mm at 70 kHz, ≥2.8 mm at 200 kHz), whereas the theoretical TS of the resonance model was similar to the experimental TS of small vesicles (≤3.1 mm at 70 kHz, ≤1.9 mm at 200 kHz). Finally, we estimated TS-size equations according to the gas-size relationships and compared the results.