We report the application of DNA methylation immunoprecipitation coupled with de novo high throughput sequencing for profiling and identifying methylation alterations in liver tumours of un-sequenced flatfish dab (Limanda limanda). We achieved approximately 90 million and 88 million paired-end reads for hepatocellular adenoma (HCA) and corresponding healthy surrounding tissue (S.T) respectively, of which 47,052,820 and 45,008,483 were uniquely mapped. By calculating the fold changes observed between HCA and S.T we identified 1693 CpG island containing regions that are differentially methylated in HCA compared to S.T (fold change >1.5). The identified genes were further annotated for gene name and biological functions. Ingenuity Pathway Analysis demonstrated enrichment of networks and top functions, such as cell-to-cell signalling, cell cycle, DNA replication, cellular assembly and organisation as well as canonical pathways including Wnt/β-catenin signalling, growth hormone signalling and apoptosis signalling associated with the differentially methylated genes in HCA compared to S.T. Finally, our data showed that MeDIP-HTP sequencing achieves substantial overlapping reads for construction of contigs and annotation of the based on closely related species for studying DNA methylation in un-sequenced species. Overall design: Investigating alteration in methylation profile of heaptocellular adenoma compared to healthy surrounding tissue of flatfish dab.