Abstract: The purpose of this study was to evaluate tshe optimum number of spermatozoa for highest fertility in Ross-308 broiler breeder chicken. About 24 Ross hens in 4 treatment groups were used. Semen was collected from 8 Ross roosters by abdominal massage and pooled in glass tube. After initially dilution with modified Ringer’s solution and determination the spermatozoa concentration by hemocytometer, the semen was diluted further. About 50, 75, 100 and 150 million spermatozoa in 0.1 mL were inseminated to treatment groups for 4 weeks (4 times repeating). Eggs were collected daily from the 2nd day post insemination and incubated. After 7 days of incubation, the eggs were candled for fertility rate. In treatment groups that were inseminated with 50, 75, 100 and 150 million spermatozoa, fertility rates were 52.85±4.15, 72.37±5.28, 87.64±4.81 and 89.12±3.74%, respectively. As shown to this results, fertility rate in treatment group that was inseminated with 50 million spermatozoa was lowest (52.85±4.15%) and then increased significantly (p<0.05) with increasing the sperm number and reached to maximum in treatment group that was inseminated with 100 million spermatozoa (87.64±4.81%). Difference of fertility rate between treatment groups that were inseminated with 100 and 150 million spermatozoa (87.64±4.81 and 89.12±3.74%, respectively) was not significant (p>0.05). It is concluded that optimum dose of spermatozoa for artificial insemination in Ross-308 chicken was 100 million sperm.