Semen was collected from Rhode Island Red and White Leghorn roosters. After adjusting the sperm number from both breeds, half of the semen was frozen-thawed in the presence of glycerol. Frozen and unfrozen semen from both breeds was mixed 1:1 with all four possible combinations and inseminated into Rhode Island Red hens. Feather colour of chicks was used to determine which breed fertilized the eggs. Results showed that sperm cells retained 19.7% [95% confidence interval = (12.8, 30.4)] of the relative fertilizing ability after freezing. Furthermore, Rhode Island Red spermatozoa had 1.5 times [95% confidence interval = (1.1, 2.0)] higher relative fertilizing ability than did White Leghorn spermatozoa. The heterospermic competition assay method is a powerful tool for estimating the relative fertilizing ability of the sperm cells.