Tuna fisheries support one of the world's most valuable markets, with over 50% of the catch coming from drifting fish aggregating devices (DFADs). To locate and quantify tuna on DFADs, fishermen mostly use acoustic technologies, which significantly reduce the nominal fishing effort, especially in tropical purse seine fisheries. However, to date, discrimination between species using purely acoustic methods has not been refined due to a lack of information on the acoustic response of each species at different frequencies. Three tuna species can be found simultaneously at DFADs: skipjack or SKJ (Katsuwanus pelamis), bigeye or BET (Thunnus obesus), and yellowfin or YFT (Thunnus albacares), of which only the acoustic frequency responses of SKJ and BET have been published. In this study, we present the frequency response obtained from ex situ measurements of YFT recorded at 38, 70, 120 and 200 kHz. Records based on two data sets were used to describe the relationship between acoustic signal or target strength (TS; dB re 1m2) and fish length across frequencies. The results described a flat response across frequencies, with b20 (standard deviation) values of -72.4 (9), -73.2 (8), -72.3 (8), and -72.3 (9) dB at 38, 70, 120, and 200 kHz, respectively. These results, combined with previously published increasing (SKJ) and decreasing (BET) responses, were used to develop a discrimination algorithm for these 3 species. The algorithm was tested using acoustic data and catches from commercial campaigns aboard a tuna vessel.