Toll-like receptors (TLRs) have emerged as crucial sensors of invading microbes through recognition of pathogen-associated molecular patterns (PAMPs) in viruses, bacteria, fungi and protozoa. The polymorphisms in TLRs are closely associated with the resistance to pathogen infections. TLR3 involved in the recognition of double stranded RNA in humans, mice, pigs and fishes. In present study, the nucleotide sequence polymorphisms of TLR3 gene in grass carp (Ctenopharyngodon idella) (CiTLR3) were investigated to explore their association with susceptibility/resistance to grass carp reovirus (GCRV). Twelve single nucleotide polymorphisms (SNPs) and an ins/del mutation were detected in the complete sequence of CiTLR3. Ten of them were sited in the non-coding region. The two SNPs in exon were synonymous mutation. The ins/del mutation was coincidental at the start codon. To investigate the association between the polymorphism and the susceptibility/resistance to GCRV, we selected eight SNPs in the noncoding region and analyzed the genotype and allele distribution in susceptible and resistant groups with PCR-RFLP. The statistical results indicated that only 764 G/T was significantly associated with the resistance of grass carp to GCRV both in genotype (P¼ 0.040) and allele (P¼ 0.025). Linkage disequilibrium analysis revealed 543 A/G, 488 G/T, 4116 G/T and 4731 C/T were linkage disequilibrium, and haplotype analysis revealed that haplotype GTTT frequency in susceptible group was significantly higher than that in the resistant group (OR ¼ 2.01, 95% CI 0.996e4.043, P¼ 0.049). To further confirm the correlation, an additional infection experiment was carried out. The mortality in the 764 GG genotype individuals was significantly lower than GT genotype (OR ¼ 0.208, 95% CI 0.067e0.643, P¼ 0.011) and TT genotype (OR ¼ 0.183, 95% CI 0.052e0.648, P ¼ 0.015). All the results indicated that haplotype GTTT and genotype 764 TT and 764 GT individuals were susceptible to GCRV while 764 GG was resistant, which could be the optional markers for selective breeding for the GCRV-resistant grass carp n future.