Using MethylRAD-Seq method, we analyzed DNA methylation distribution in the genome of Misgurnus anguillicaudatus and compared DNA methylation variation between the diploids and tetraploids. In total, 302 111 684 MethyRAD sequence tags were obtained and mapped to the reference genome. The results showed that, the DNA methylated sites distributed mainly in gene body, followed by intron and intergenic, but fewer methylated sites were found in other functional elements. Total DNA methylation level in the tetraploids was higher than that in the diploids, especially significant differences (P＜0.01) of DNA methylation level existed in 1st Exon and upstream 200-1 500 bp region of the transcription start site (TSS 1500). However, in the promoter region, DNA methylation level in the tetraploids was lower than that in the diploids. For DNA methylation level of each sites, 1 268 CmCGG and 14 CmCWGG sites showed significant difference between the diploids and tetraploids, which mainly distributed in intron, gene body and intergenic. A total of 684 genes exhibited significant methylation variations between ploidy. KEGG Pathway enrichment analysis showed that, the genes with DNA methylation variation between ploidy enriched mainly in the growth development, immunity and mismatch repair related signaling pathways.