Mature male and female rainbow trout (Oncorhynchus mykiss) were used to study the effect of different concentrations of streptomycin in a cryopreservation solution on the sperm motility, motility duration, and fertilization rate of rainbow trout. 300mM glucose and 10% DMSO containing diluent was used as a cryopreservation solution. Three different concentrations (3600, 7200 and 10800 μg/ml) of streptomycin were added to the diluent. Our study showed that various concentrations of streptomycin in cryopreservation solution caused a significant reduction in the percentage and duration of rainbow trout spermatozoa motility after freezing-thawing. The percentage of egg fertilization also decreased. When streptomycin-containing groups were compared with each other, a statistical difference was found (p<0.05). However, no statistical difference was observed in terms of the percentage of eyed-embryos.