In the present study, the potential of serological methods, the repetitive extragenic palindromic PCR (REP-PCR) and the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) for the typing of the species Tenacibaculum maritimum, Tenacibaculum soleae and Tenacibaculum discolor was evaluated. Moreover, molecular and proteomic techniques were used to assess variability among strains belonging to different serotypes, as well as isolated from different host species and geographical areas. Slide agglutination and dot-blot assays demonstrated the lack of immunological relationships among Tenacibaculum species analyzed. The serotype O1 was predominant within T. maritimum isolates regardless of the fish species or geographical area. Two serotypes were distinguished within T. soleae isolates and at least one within T. discolor strains. Species- and strain-specific profiles were obtained from the analysis of T. maritimum, T. soleae and T. discolor by REP-PCR and ERIC-PCR, demonstrating their potential as diagnostic tools. The genotyping analysis using both techniques showed genetic variability among the strains of each fish pathogenic Tenacibaculum species analysed. However, Tenacibaculum strains isolated from different host species or geographical areas or belonging to different serotypes produced REP and ERIC profiles with high similarity. Analysis by MALDI-TOF-MS of the T. maritimum strains could not detect any serotype-identifying biomarkers. Serotype-specific mass peaks were found for the serotypes O1 and O2 of T. soleae and for the serotype O1 of T. discolor. However, no relationships between the proteomic profiles and the source of isolation of the strains were obtained for any of the Tenacibaculum species analysed in this study.