High-throughput sequencing is increasingly being used in combination with bisulfite (BS) assays to study DNA methylation at nucleotide resolution. Although several programmes provide genome-wide alignment of BS-treated reads, the resulting information is not readily interpretable and often requires further bioinformatic steps for meaningful analysis. Current post-alignment BS-sequencing programmes are generally focused on the gene-specific level, a restrictive feature when analysis in the non-coding regions, such as enhancers and intergenic microRNAs, is required. Here, we present Genome Bisulfite Sequencing Analyser (GBSA-http://ctrad-csi.nus.edu.sg/gbsa), a free open-source software capable of analysing whole-genome bisulfite sequencing data with either a gene-centric or gene-independent focus. Through analysis of the largest published data sets to date, we demonstrate GBSA's features in providing sequencing quality assessment, methylation scoring, functional data management and visualization of genomic methylation at nucleotide resolution. Additionally, we show that GBSA's output can be easily integrated with other high-throughput sequencing data, such as RNA-Seq or ChIP-seq, to elucidate the role of methylated intergenic regions in gene regulation. In essence, GBSA allows an investigator to explore not only known loci but also all the genomic regions, for which methylation studies could lead to the discovery of new regulatory mechanisms.