The maintenance of the endangered Caspian brown trout Salmo trutta caspius populations in Iran depends on its artificial breeding. There is no information on how current hatchery breeding protocol influences effective population size of breeders (Ne), which is a critical parameter to maintain genetic diversity in future generations. The current hatchery protocol (CHP) was comparatively evaluated with a balanced hatchery protocol (BHP), where mixtures of equal amounts of gametes per male and female breeder were used to balance parental contributions to progeny. To evaluate putative differences in viability between families, individual crosses were performed and fertilized ova of different families were mixed to constitute balanced family pools. 1440 alevins were totally sampled in the crosses performed from the 11 breeders. An exclusion-based parentage approach using three polymorphic microsatellite markers unambiguously assigned more than 93% of progeny to a single pair of parents. Significantly different contributions of breeders to progeny were observed in CHP (p < 0.05). The primary constraint on Ne in BHP was the unbalanced contribution of males, which seemed a consequence of sperm competition in mixed fertilization caused by differences in sperm quality. Sperm motility duration was positively correlated with the number of sired progeny by each male. The results illustrate the limitations of the BHP in minimizing the loss of genetic diversity observed in CHP. A protocol based on mixture of equal number of fertilized ova from individual male × female crosses emerged as the best alternative for conservation of Caspian brown trout.