All-female progeny is preferable in the reproduction of rainbow trout. Using sex-reversed (SR) female rainbow trout by steroid hormones as broodstock in the reproduction is the most appropriate way to all-female progeny. This study compared mature SR female rainbow trout by ethisterone (ET), 11 beta-Hydroxyandrostenedione (HD) and 17 alpha-Methyltestosterone (MT) in terms of spermatological parameters and health status, for the first time. Spermatozoa concentration, motility characteristics, seminal plasma pH, testosterone and gonadotropins in seminal plasma and blood serum, blood parameters of SR females were evaluated, comparing with testicular and stripped milt, and blood from normal males (NM). The SR females by HD had greater gonadosomatic index and better motility characteristics compared with other groups. Also, blood parameters of this group were more similar to those in NM. The SR females by ET had the lowest haematocrit and the highest glucose and activities of the enzymes compared with the other groups. The measured hormones were significantly the lowest in both seminal plasma and serum of the ET group. These findings suggested that using of HD for obtaining SR female should be preferred due to having the better quality spermatozoa and health status.