Hybridization and polyploidization may lead to divergence in adaptation and boost speciation in angiosperms and some lower animals. Epigenetic change plays a significant role in the formation and adaptation of polyploidy. Studies of the effects of methylation on genomic recombination and gene expression in allopolyploid plants have achieved good progress. However, relevant advances in polyploid animals have been relatively slower. In the present study, we used the bisexual, fertile, genetically stable allotetraploid generated by hybridization of Carassius auratus red var. and Cyprinus carpio L. to investigate cytosine methylation level using methylation-sensitive amplification polymorphism (MSAP) analysis. We observed 38.31% of the methylation changes in the allotetraploid compared with the parents at 355 randomly selected CCGG sites. In terms of methylation status, these results indicate that the level of methylation modification in the allotetraploid may have increased relative to that in the parents. We also found that the major methylation changes were hypermethylation on some genomic fragments and genes related to metabolism or cell cycle regulation. These results provide circumstantial evidence that DNA methylation might be related to the gene expression and phenotype variation in allotetraploid hybrids. Our study partly fulfils the need for epigenetic research in polyploid animals, and provides evidence for the epigenetic regulation of allopolyploids.