Little is known about how patterns of DNA methylation change during mammalian spermatogenesis. 5hmC has been recognized as a stable intermediate of DNA demethylation with potential regulatory functions in the mammalian genome. However, its global pattern in germ cells has yet to be addressed. Here, we first conducted absolute quantification of 5hmC in eight consecutive types of mouse spermatogenic cells using liquid chromatography-tandem mass spectrometry, and then mapped its distributions in various genomic regions using our chemical labeling and enrichment method coupled with deep sequencing. We found that 5hmC mapped differentially to and changed dynamically in genomic regions related to expression regulation of protein-coding genes, piRNA precursor genes and repetitive elements. Moreover, 5hmC content correlated with the levels of various transcripts quantified by RNA-seq. These results suggest that the highly ordered alterations of 5hmC in the mouse genome are potentially crucial for the differentiation of spermatogenic cells.