Single-cell genome-wide bisulfite sequencing (scBS-Seq) is a method that accurately measures DNA methylation at up to 48.4% of CpGs. This technique allows for the assessment of 5mC heterogeneity within cell populations across the entire genome. The study reports scBS-Seq as a versatile tool for exploring DNA methylation in rare cells and heterogeneous populations. DNA methylation (5mC) is an epigenetic mark with critical roles in regulation and maintenance of cell-type-specific transcriptional programs. The development of bisulfite sequencing (BS-Seq) has revolutionized our understanding of 5mC functionality by offering single cytosine resolution and absolute quantification of 5mC levels genome-wide. Recent advances have demonstrated the power of single-cell sequencing analyses for the deconvolution of mixed cell populations. Incorporation of epigenetic information into this single-cell arsenal will transform our understanding of gene regulation and reveal new insights into epigenetic heterogeneity. The study describes a modified version of Post-Bisulfite Adaptor Tagging (PBAT) for scBS-Seq, where bisulfite treatment is performed first, resulting in simultaneous DNA fragmentation and conversion of unmethylated cytosines. This method allows for the generation of multiple copies of each fragment through five rounds of random priming and extension. The study performed scBS-Seq on metaphase-II (ovulated) oocytes (MIIs) and mouse embryonic stem cells (ESCs) cultured either in 2i or serum conditions. MIIs are an excellent model for technical assessment due to their high homogeneity and distinct DNA methylome. ESCs grown in serum conditions exist in a state of dynamic equilibrium characterized by transcriptional heterogeneity. The study found that both 2i and serum ESCs exhibited 5mC heterogeneity. scBS-Seq was able to reveal DNA methylation heterogeneity at the single-cell level. The study also demonstrated the reproducibility and accuracy of scBS-Seq. Low levels of non-CpG methylation across all samples revealed a minimum bisulfite conversion efficiency of 97.7%. CpG sites in MIIs were overwhelmingly called methylated or unmethylated, consistent with a highly digitized output from single cells. The study found that scBS-Seq provides information on all genomic contexts, including regulatory regions. The ability to capture the DNA methylome from individual cells is critical for a full understanding of early embryonic development, cancer progression, and induced pluripotent stem cell (iPSC) generation. In summary, the study provides a proof-of-principle that large-scale single-cell epigenetic analysis is achievable, and demonstrates that scBS-Seq is a unique and powerful approach to accurately measure 5mC across the genome of single cells and to reveal 5mC heterogeneity within cell populations.Single-cell genome-wide bisulfite sequencing (scBS-Seq) is a method that accurately measures DNA methylation at up to 48.4% of CpGs. This technique allows for the assessment of 5mC heterogeneity within cell populations across the entire genome. The study reports scBS-Seq as a versatile tool for exploring DNA methylation in rare cells and heterogeneous populations. DNA methylation (5mC) is an epigenetic mark with critical roles in regulation and maintenance of cell-type-specific transcriptional programs. The development of bisulfite sequencing (BS-Seq) has revolutionized our understanding of 5mC functionality by offering single cytosine resolution and absolute quantification of 5mC levels genome-wide. Recent advances have demonstrated the power of single-cell sequencing analyses for the deconvolution of mixed cell populations. Incorporation of epigenetic information into this single-cell arsenal will transform our understanding of gene regulation and reveal new insights into epigenetic heterogeneity. The study describes a modified version of Post-Bisulfite Adaptor Tagging (PBAT) for scBS-Seq, where bisulfite treatment is performed first, resulting in simultaneous DNA fragmentation and conversion of unmethylated cytosines. This method allows for the generation of multiple copies of each fragment through five rounds of random priming and extension. The study performed scBS-Seq on metaphase-II (ovulated) oocytes (MIIs) and mouse embryonic stem cells (ESCs) cultured either in 2i or serum conditions. MIIs are an excellent model for technical assessment due to their high homogeneity and distinct DNA methylome. ESCs grown in serum conditions exist in a state of dynamic equilibrium characterized by transcriptional heterogeneity. The study found that both 2i and serum ESCs exhibited 5mC heterogeneity. scBS-Seq was able to reveal DNA methylation heterogeneity at the single-cell level. The study also demonstrated the reproducibility and accuracy of scBS-Seq. Low levels of non-CpG methylation across all samples revealed a minimum bisulfite conversion efficiency of 97.7%. CpG sites in MIIs were overwhelmingly called methylated or unmethylated, consistent with a highly digitized output from single cells. The study found that scBS-Seq provides information on all genomic contexts, including regulatory regions. The ability to capture the DNA methylome from individual cells is critical for a full understanding of early embryonic development, cancer progression, and induced pluripotent stem cell (iPSC) generation. In summary, the study provides a proof-of-principle that large-scale single-cell epigenetic analysis is achievable, and demonstrates that scBS-Seq is a unique and powerful approach to accurately measure 5mC across the genome of single cells and to reveal 5mC heterogeneity within cell populations.