The hypothalamic gonadotropin-releasing hormone receptor (GnRHR) plays an essential physiological role in reproductive function, which triggers the synthesis and release of luteinizing hormone and follicle stimulating hormone in the pituitary. The objective of this study was to investigate the effects of polymorphisms of GnRHR gene on the quality of fresh and frozen semen in Holstein bulls. The PCR-RFLP method was applied to detect G286A and T340C transitions determining MboII and BspMI polymorphisms, respectively, in the exon I of bovine GnRHR gene and evaluated its associations with sperm quality traits in 131 Holstein bulls. In polymorphic locus 286, bulls with the GA genotype had significantly higher sperm motility in frozen semen (FMOT) than GG genotype (P < 0.01). In polymorphic locus 340, bulls with heterozygote CT genotype had significantly higher sperm motility (MOT), semen volume per ejaculate (VOL), and lower abnormal spermatozoa rate (ASR) than homozygote TT genotype (P < 0.05). Bulls contained one A allele or C allele had a favorable, positive effect on sperm quality traits. These results indicate that GnRHR gene can be a potential marker for improving sperm quality traits, and imply that bulls with GA or CT genotype should be selected in breeding program.