Strategy of managing antibiotic-resistant Vibrio alginolyticus, a bacterial pathogen that threatens human health and animal farming, is not available due to the lack of knowledge about the underlying mechanism of antibiotic resistance. Here, we showed that gentamicin-resistant V. alginolyticus(VA-RGEN) has four mutations on metabolism and one mutation on a two-component system by whole-genome and PCR-based sequencing.