DNA methylation in intragenic regions plays a conserved role in regulating alternative promoters. This study investigates the function of intragenic DNA methylation in human and mouse tissues, revealing that it is more prevalent in intragenic and intergenic regions than in 5' promoters. The methylation status of CpG islands (CGIs) in these regions overlaps with RNA markers of transcription initiation and trimethylation of H3K4, a histone modification enriched at promoters. The general and CGI-specific patterns of methylation are conserved in mouse tissues. Analysis of the SHANK3 locus in humans and its mouse homologue shows that tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies. The study also highlights the complexity of DNA methylation-associated regulation of alternative promoters within gene bodies, including differences in this regulation within a single cell type from distinct brain regions, and in different regions of the same gene in the same cell. The integration of epigenomics and comparative genomics provides new insights into the role of DNA methylation in gene expression during development and cancer.DNA methylation in intragenic regions plays a conserved role in regulating alternative promoters. This study investigates the function of intragenic DNA methylation in human and mouse tissues, revealing that it is more prevalent in intragenic and intergenic regions than in 5' promoters. The methylation status of CpG islands (CGIs) in these regions overlaps with RNA markers of transcription initiation and trimethylation of H3K4, a histone modification enriched at promoters. The general and CGI-specific patterns of methylation are conserved in mouse tissues. Analysis of the SHANK3 locus in humans and its mouse homologue shows that tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies. The study also highlights the complexity of DNA methylation-associated regulation of alternative promoters within gene bodies, including differences in this regulation within a single cell type from distinct brain regions, and in different regions of the same gene in the same cell. The integration of epigenomics and comparative genomics provides new insights into the role of DNA methylation in gene expression during development and cancer.