Streptococcosis/lactococcosis is a hyperacute systemic disease that can occur in marine and fresh waters of many species of fish. The aim of this work was to study the disease outbreak in the major rainbow trout (Oncorhynchus mykiss) production of Iran.108 Gram positive cocci isolates were obtained from diseased trout in seven provinces with major trout production during 2008 till 2009. These bacterial isolates were characterized using phenotypic and molecular studies. The isolates were also analysed phylogeneticaly and compared with the available data.49 samples (45.37%) were identified as Streptococcus iniae, 37 samples (35.2%) matched with Lactococcus garvieae; and 22 samples (19.43%) were identified as members of Streptooccus genus by culture-based and biochemical tests of API 50 CH, API 20 STREP and rapid 32 STREP systems. Using universal primers for differentiation of Streptococcus sp. and Enterococcus sp, all 108 samples were identified as Streptococcus sp. with a target region of 500 bp. Single specific PCR resulted in identification of 64 (59.2%) isolates as S. iniae and 44 (40.8%) isolates as L. garvieae. The phylogenetic analysis of the S. iniae isolates resulted in maximal similarity to some strains reported from Taiwan and to all Brazilian strains. Also, one strain showed less sequence similarity values with other tested strains although this strain has high similarity with ATCC 29178 strain, all reported Chinese, and some Taiwanian strains. Also, analysis of S. iniae LctO gene sequence showed that this isolate clustered within the S. iniae group. The sequence analysis of L. garvieae strains also showed that they have maximum similarity to all Japanese and Chinese strains, but one strain has lower sequence similarity values with all other recorded strains.[corrected] The results of this study clearly show that trout farming in Iran is severely affected by both species of S. Iniae and L. garvieae and requires serious preventive criteria.