Chromatin structure plays an important role in the regulation of gene expression. Methylation of lysine residues on histone tails is an epigenetic mark that influences chromatin repression when specifically imparted on lysines 9 and 27 of histone H3, and on lysine 20 of H4. Histone lysines can be mono-, di-, and trimethylated, and all three modification states have been identified in different nuclear domains. Correlation of these methylated histone states to different stages of cell differentiation, however, is not extensive. Mammalian spermatogenesis is a developmental process ideal for studying the epigenetic control of differentiation. Maintenance of spermatogonial stem cells requires the transcriptional repressor Plzf, but a role for histone methylation has not been established. Here we show that Plzf-expressing spermatogonia completely lack monomethyl-H3-K27 and monomethyl-H4-K20, and contain very little monomethyl-H3-K9. Dimethylated H3-K27 and H4-K20 are detected as punctate foci in Plzf-positive cells, but dimethyl-H3-K9 is absent. Trimethylated H3-K9 and H4-K20 exhibit a unique perinuclear distribution that coincides with Plzf expression, localizing to punctate foci in more differentiated spermatogonia. Loss of Plzf correlates with increased punctate distribution of trimethylated H3-K9 and H4-K20 at the expense of perinuclear localization. These data signify the possible importance of different histone lysine methylation states in the epigenetic control of spermatogenesis.