Aim: The objectives of our study is to determinate the antibiotic susceptibility of this organism to different antibiotics to determine the discriminatory power of the molecular typing methods. Methods and Results: In this study, 50 Photobacterium damselae subsp. damselae isolates from Scomber australasicus and Rachycentron canadum were collected in Taiwan and their resistance to 15 different antimicrobial agents was determined. In addition, random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrolysis (PFGE) were performed to study the epidemiology and clonal relationship of P. damselae subsp. damselae. The results showed that the 50 isolates generated 25 typeable profiles with multidrug resistance to 3-7 antimicrobials. The results also indicate that the RAPD and PFGE methods have high discriminatory power for molecular subtyping. Conclusion: Photobacterium damselae subsp. damselae isolates from fish to examine for multidrug resistance to antimicrobials. RAPD and PFGE methods revealed the high discriminatory power for molecular subtyping and provided information that could be used for risk assessment of P. damselae subsp. damselae infections. Significance and Impact of the Study: These results may help in epidemiological investigations of P. damselae subsp. damselae and may be useful in controlling or treating P. damselae subsp. damselae infections in aquaculture and clinical therapy.