This study aimed to investigate the effect of the Rainbow trout seminal plasma (RTSP) supplemented (control, 10% or 15%) lyophilized extender on freezability of ram semen. Collected semen was pooled and split into two aliquots, and each of the ejaculates was diluted with fresh or lyophilized extenders with RTSP (0%, FC and LC; 10% F10 and L10or 15% F15 and L15) using two-step dilution method. Semen was frozen using the programmable freezing machine. Semen samples were examined for sperm motility, defective acrosomes, plasma membrane integrity and DNA fragmentation at native and post-thaw stage. The highest percentages of post-thaw motility and plasma integrity were observed in the F10, F15, and L15 (P 0.05). There was no significant difference in the rate of post-thaw defective acrosomes when the other extender groups were compared with the control (P 0.05). The highest percentage of post-thaw DNA fragmentation values were observed in the FC group, while the lowest DNA fragmentation was obtained in the F15 but only significant different from FC and LC groups. In conclusion, the findings of this study show that the lyophilized extender with 15% RTSP added can be used successfully for freezing ram semen.