Olfaction plays an important role in the survival and reproduction of fish, as it can perceive and identify thousands of different odor molecules, which help fish to carry out life activities such as feeding, reproduction, migration and avoidance of enemies. The ability of V1Rs to recognize pheromones plays an important role in animal reproduction and mate recognition. To investigate the differences in the structure of olfactory epithelium (OE) and the evolution of vomeronasal type-1 receptors (V1Rs) in different fish species, this study compared the structural characteristics of the OE of nine fish species based on histological studies. The sequence, protein structure and evolution of the V1Rs family in these nine fish species were identified and analyzed using comparative genomics and bioinformatics. Additionally, tissue expression patterns of the V1Rs in male and female Misgurnus anguillicaudatus were investigated by qRT-PCR. The results revealed that six species of Cypriniform fish have G-type olfactory plates types, while Siluriform fish have H-type, and Perciform fish have both F-type and G-type. There is a significant variation in the number of olfactory plates among the nine fish species studied, for example, Micropterus salmoides has only seven olfactory plates while Pelteobagrus fulvidraco has eighty. The study identified a total of 58 complete V1Rs genes, divided into six subtypes (V1R1, V1R2, V1R3, V1R4, V1R5, V1R6). Each of the nine fish species genomes contains one V1R1 and one V1R2 gene; however, the genome of the Culter alburnus lacks V1R3 and V1R4 genes, while the Siniperca chuatsi is missing the V1R5 gene. Additionally, the Megalobrama amblycephala, P. fulvidraco and C. alburnus each have two copies of the V1R5 gene. The results of qRT-PCR showed that all seven V1Rs genes were significantly highly expressed in the olfactory epithelium (OE) of both female and male M. anguillicaudatus compared with the control group (muscle tissue) (P<0.05), except for V1R3b which did not show significant expression differences between OE and muscle tissue. Furthermore, there was lower expression in the brain and beard tissues with no significant difference compared to the control group (P>0.05). However, there was a significant difference in gonadal tissues between male and female M. anguillicaudatus. Specifically, V1R1, V1R4, V1R5 and V1R6 were significantly higher in the testis than in the ovary, whereas the V1R3b was significantly higher in ovary than in testis, V1R2 and V1R3a were not significantly different in male and female M. anguillicaudatus. This study not only elucidates the morphological and histological structure of OE and the evolutionary differences of V1Rs in different fishes, but also provides a foundation for further investigation into the mechanisms and roles of fish olfactory mate recognition and the functional aspects of V1Rs genes.