Background: The enhanced auditory abilities of certain fish are dependent on specialized hearing structures. Several gas-holding structures, including the suprabranchial chamber, otic gas bladder, and Weberian apparatus-linked gas bladder, have been demonstrated to improve the hearing ability of fish. The walking catfish (Clarias batrachus), a benthic species, is unique in that it has both a suprabranchial chamber (SC) and a Weberian apparatus-linked, encapsulated gas bladder (WGB). This study aimed to investigate the respective roles of these two structures in contributing to the overall hearing ability of walking catfish. Results: The auditory evoked potentials method was used to measure hearing thresholds in intact fish and fish with a deflated WGB and/or SC. Gas removal from the WGB increased auditory thresholds at all frequencies tested (0.4 to 5.0 kHz), whereas injection of water into the SC shifted the threshold only at frequencies below 3.0 kHz. However, such a correlation was not observed for the SC at 4.0 and 5.0 kHz.