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DOI : 10.17693/yunusae.v16i26717.281024 Viewed : 3171 - Downloaded : 1833 In this study, the effects of three different dosages of grape molasses (5, 7, 10 %) as a diluent were investigated on post‐thaw motility, sperm viability and fertilization rate of frozen spermatozoa from the rainbow trout (Oncorhynchus mykiss). Semen from six male fish sperm was diluted with in extenders containing 5, 7, and 10% grape molasses with dimethyl sulfoxide 7%. The semen was frozen in 0.25 ml straws in liquid nitrogen vapor and stored in liquid nitrogen for 2 weeks. From the point of grape molasses effect over sperm motility, grape molasses level of 10 % was found more beneficial (P<0.001), and there was no significant difference between various level of grape molasses on fertilization rate (P>0.05). But, the 10% grape molasses solution was better (96.4%) than the glucose solution which has the highest fertilization rate (P<0.05). As a result of the study, the level of 10% grape molasses is given that might be used to freezing rainbow trout sperm. Keywords : Aquaculture,freezing,cryopreservation,milt