Interferon-β promoter stimulator 1 (IPS-1) plays a pivotal role in the production of type I interferon (IFN) and pro-inflammatory cytokines. Though its function in innate immunity was widely studied, its genetic polymorphisms and evolution information were rarely known. Herein, the present study firstly identified and characterized the genomic organization of CiIPS-1 (IPS-1 gene of grass carp, Ctenopharyngodon idella) and its nucleotide polymorphisms. CiIPS-1 consists of 8789 bp, with a 314 bp 5′–flanking region, 6 exons and 5 introns. After a challenge experiment, statistical analysis was performed to assess the association between the 17 polymorphisms of CiIPS-1 and the natural resistance of C. idella to grass carp reovirus (GCRV), which revealed that –3741 C/T in the intron 1, 933 C/G in the coding sequence and 2299 G/T in the last intron were significant (P < 0.05). The subsequent secondary challenge test further confirmed that –3741TT group were more resistant than –3741CC group to GCRV infection, while 933GG and 2299GG stocks were respectively more susceptible than 933CC and 2299TT stocks (P < 0.05). In addition, haplotype and polymorphism–polymorphism interaction analyses uncovered four significant haplotypes (P < 0.05) and two notable polymorphisms. These findings may provide basic data for the further functional research of CiIPS-1, and genetic markers for molecular and transgenic breeding of C. idella.