Fish pasteurellosis is an infectious disease that affects several teleost species living in temperate marine waters. The pathogen responsible, Photobacterium damselae subspecies piscicida, shows high genetic similarity with P. damselae subsp. damselae, making subspecies discrimination extremely laborious. Here we report for the first time a PCR-RFLP method for the identification of P. damselae subsp. piscicida without prior isolation in pure culture. Genomic sequence information was obtained through cloning and sequencing of RAPD products. Two P. damselae-specific primer pairs were developed and tested on 17 strains of P. damselae subsp. piscicida, 10 strains of P. damselae subsp. damselae, and 6 closely related control species. High sensitivity was achieved in PCR amplification on serially diluted samples (<180 fg of pure bacterial DNA or <10 fg, depending on the amplified fragment). Restriction analysis of PCR products showed a unique digestion profile for all P. damselae subsp. piscicida strains. The same PCR-RFLP method was implemented on total DNA samples extracted from experimentally infected sea bream and sea bass. Positive results were obtained on fish with clear signs of the disease as well as on challenged, but asymptomatic, fish. The method presented here might provide a useful tool for both prevention and rapid diagnosis of fish pasteurellosis.