Aims To investigate the presence of prophage in Streptococcus iniae, a highly problematic fish pathogen. Methods and Results Cross-spotting assays and mitomycin C inductions were conducted to screen for prophage in 48 Strep.iniae isolates. Bacteriophages were characterized by plaque assays, transmission electron microscopy and DNA restriction enzyme digestion. Plaque assays confirmed prophages in 14 center dot 6% of isolates. Phages vB_SinS-44, vB_SinS-45, vB_SinS-46 and vB_SinS-48 lysed 78 center dot 5% of Strep.iniae isolates and displayed distinctive host ranges. Microscopy revealed virions exhibiting long, non-contractile tails and isometric heads consistent with phages from the family Siphoviridae. Restriction digests revealed genome sizes ranging from 27 center dot 5 to 66 center dot 3kbp, with distinct cutting patterns that indicate the presence of related prophages in bacteria isolated from different geographic regions. Conclusions The rate of prophage carriage found is comparably low and induction rates varied between phages. The four characterized Siphoviridae phages have broad host ranges within the Strep.iniae isolates. Significance and Impact of the Study This is the first description and characterization of lysogenic phages from Strep.iniae. These phages are candidates for research and diagnosis of the bacterium and their identification should accelerate the discovery of lytic phages to be trialled against Strep.iniae infections in fish.