BACKGROUND: Strepotococcosis caused by Streptococcus iniae is one of the important emerging bacterial diseases in aquaculture sector worldwide. ObjectiveS: In this study, the genetic diversity of S. iniae strains was assessed in some rainbow trout farms in Iran. Methods: Gram positive and catalase negative bacterial isolates were first obtained from 100 trout fish farms in 8 states using routine bacteriological and molecular (PCR) works. The genetic diversity of these bacterial isolates was then assessed using restriction fragment length polymorphism (RFLP) method. Results: Seventy-seven strains of Gram positive and catalase negative cocci were isolated from diseased trout. PCR analysis resulted in identification of 27 strains as S. iniae. RFLP analysis of these strains using 9 digestive enzymes resulted in production of 29 bands with different molecular weight (62-940bp).  Phylogenetic relationship of these strains grouped them in two distinct clusters. Twenty-six strains from Tehran, Mazandaran, Gilan, Lorstan, Fars and Charmaha-va-Bakhtiary provinces showed high homogeneous similarity above 99%, while one strain from Mazandaran province showed some differences with other strains. ConclusionS: S. iniae isolates in trout aquaculture in Iran possess  low genetic diversity.