Biomonitoring is the main way to assess the pollution status in aquatic ecosystems where Biomarkers response is used at different levels of trace contaminants on the aquatic organisms. Recently, many investigations have been focused to the genotoxic biomarker in aquatic ecosystems owing to mutagen effects of the contaminant on such environment. However, one of the fundamental problems in this area is the lack of proper maintenance protocol for transferring of biological samples with minimum damage to the laboratory. For this purpose, it is required to use appropriate conditions evaluation and standardization to avoid any possible test error analysis of the habitat situation in this matter. In this study, the Comet assay as a new method in Biomarker studies at the molecular level that is based on measuring damages reflected as strand breakage, was established to investigate the effects of three storage solutions (FBS, L-15 and PBS) on DNA integrity in red blood cell of rainbow trout (Oncorhynchus mykiss) at different times after blood sampling (6, 12, 24 and 48 h). Cell viability as well as was determined by trypan blue dye exclusion in time intervals. The Results of Comet assay in blood samples which has been maintained in L-15 and FBS solutions, did not exhibit significant differences in DNA damage between different times, while samples preserved 24 h in PBS indicated the significant increase in the levels of DNA damage compared to time 0 as control (P< 0.05). According to the results in this study, it is appeared that media osmolarity is the most important factor in maintaining viability of red blood cells during storage in different solutions. Overall, this result indicates that the blood samples from freshwater fish can be preserved up to 48 h in FBS or L-15 mediums at 4°C in the absence of light with no significant DNA damage.