GnIH (gonadotropin inhibitory hormone) has been proven to be a hypothalamic neuropeptide that regulates the reproductive negatively, which is related to reproduction. In this study, the full-length cDNA sequences of gnih and gnihr3 genes of diploid red crucian carp and allotriploid crucian carp were obtained by PCR and RACE technology. Real-time PCR was used to analyze the expressions of gnih and gnihr3 genes in different tissues of different ploidy Cyprinids. The results showed that the expression level of gnih gene in the hypothalamus of red crucian carp was higher than that of allotriploid crucian carp; the gnihr3 gene was expressed differently on the hypothalamic–pituitary–gonad axis (HPG). Nevertheless, it was noteworthy that regardless of non-breeding or breeding season the expression of gnih in the hypothalamus from red crucian carp was higher than that of allotriploid crucian carp. Simultaneously, the expression level of gnihr3 gene in hypothalamus and pituitary of red crucian carp was higher than that of allotriploid crucian carp, but the expression of gnihr3 in the ovary of allotriploid crucian carp was higher than that of red crucian carp. Through in situ hybridization, the locations of gnih and gnihr3 in brain or pituitary of different ploidy fishes were analyzed. Strong hybridization signals of gnih could be detected in Npp, Npo, NLT in the brain of red crucian carp and allotriploid crucian carp. Strong hybridization signals of gnihr3 were located mainly in the PPD and RPD regions of the pituitary, and weak hybridization signals were distributed in NH and PI regions. Meanwhile, the signal of gnihr3 hybridization of allotriploid crucian carp in the pituitary was much weaker than that of red crucian carp. The results showed that gnih is mainly expressed in the hypothalamus, combining with the receptors distributed in HPG widely and acting through multiple pathways, which participate in the regulation of gonad development of different ploidy cyprinids through multiple ways. There are differences in the expression of gnih and gnihr3 mRNA in different ploidy fish tissues. This study provides molecular evidence for sterility of allotriploid crucian carp, which is important for fish reproduction and genetics breeding.