Aims: Intraspecific differentiation and characterization of Vibrio alginolyticus strains isolated from cultured fish in Spain. Materials and Results: Thirty-four Vibrio alginolyticus strains isolated from cultured fish were intraspecifically characterized on the basis of biochemical and exoenzymatic patterns, outer membrane protein (OMP) profiles, ribotyping and plasmid analyses. The typing methods used did not allow to group V. alginolyticus isolates on the basis of their sources of collection. A higher homogeneity was observed in OMP profiles. A high percentage of isolates were plasmidless. Ribotyping was the highest discriminatory typing method, as all the isolates tested presented 23 profiles using the HindIII restriction enzyme. On the basis of the ribotyping pattern, a similarity matrix and a dendrogram were constructed. Conclusions: The results obtained indicate that V. alginolyticus strains isolated from southwestern Spain belong to different clonal lineages. Significance and impact of the study: This study has shown differences with other similar studies carried out in other areas of Europe with strains of V. alginolyticus with respect to the clonal lineages of the strains isolated in southwestern Spain.