Fertility is one of the most important parameters in breeder farms and cockerels play an outstanding role in fertility of eggs in broiler breeder units. Todays, supplementation of feed-additives such as organic selenium is used to increase fertility. The aim of this study was to evaluate the effects of different levels of Nano-Selenium (Nano-Se) on expression of molecular markers of spermatogonial stem cells (SSCs) in testis of broiler breeder males. A total of 30 roosters of 40 weeks age were randomly divided into 5 groups. Groups were as follow: 1) control (normal diet), 2) diet supplemented with 0.3 mg/kg sodium Selenite, 3) diet supplemented with 0.15 mg/kg Nano-Se, 4) diet supplemented with 0.3 mg/kg Nano-Se and 5) diet supplemented with 0.6 mg/kg Nano-Se. At the end of experimental period, birds autopsied and samples from testis of all birds were taken. The samples were used to examine the β1-integrin (CD29), thy-1(CD90 and NANOG mRNA expression by quantitative Real-Time PCR. The results of this study showed that testis of the groups fed with diets supplemented with 0.6mg/kg and 0.15mg/kg of Nano-Se had the highest and lowest mRNA expression of SSCs markers, respectively. In conclusion, the present study indicated that Nano-Se had advantages to sodium Selenite and 0.6 mg/kg of Nano-Se supplemented in males' diet in broiler breeders farms may contributes to optimal fertility via increasing mRNA expression of SSCs markers of roosters' testis and could be used to delay reduction of fertility caused by aging in broiler breeder males.