Aquaporin 9 plays critical roles in aspects of energy homeostasis, metabolism, gluconeogenesis, fat synthesis and even the individual growth and development. So the Aquaporin 9 (AQP9) gene is a potential candidate gene for bovine growth traits. In this study, we detected the polymorphism of the bovine AQP9 gene including all exons by PCR-SSCP and DNA sequencing methods with six pairs of PCR primers in 555 individuals from three cattle breeds. Three novel SNPs (NC_007308:g.47575 C > T, 47615 C > T, 47690A > G) were detected using P6 primer. The linkage disequilibrium analysis indicated that the three SNPs were completely linked (r2 = 1), which constructed three genotypes (AA, AB, BB). The genotype AB was dominant in all three breeds. The frequencies of haplotype A and haplotype B were almost equivalent between each other. The individuals with genotype AB were significantly higher than those individuals with genotype BB in body weight (p < 0.01), chest circumference (p < 0.05) and rump length (p < 0.05). Moreover, individuals with genotype AA were significantly higher than those of individuals with genotype BB in body height (p < 0.01). These results suggested that the novel SNPs could be a perfect molecular marker for marker-assisted selection (MAS) breeding.