The aim of this work was to develop a reverse line blot hybridization (RLB) assay for the identification of five fish pathogens (Tenacibaculum soleae, Tenacibaculum maritimum, Vibrio harveyi, Photobacterium damselae and Pseudomonas baetica) of importance in marine aquaculture. Species-specific probes were designed targeting the 16S–23S intergenic spacer region (ISR) or the 23S rRNA gene. Reference and clinical strains of each target species were all correctly identified by the RLB assay, whereas all non-target strains gave unambiguous negative results with the exception of cross-reactivity between the V. harveyi probe and V. campbellii PCR product. Sensitivity limits of the RLB assay using pure cultures varied among the five target organisms from 1 to 100 pg of genomic DNA. To our knowledge, this is the first RLB protocol developed for identification of bacterial fish pathogens, and also constitutes the first molecular diagnosis tool available for P. baetica. It will be helpful for epidemiological studies and for the control of diseases in aquaculture.