Although different epigenetic marks correlate with different chromatin states, how they are integrated within single nucleosomes to generate combinatorial signals remains largely unknown. We report the successful implementation of single molecule tools constituting Fluorescence Correlation Spectroscopy (FCS), Pulse Interleave Excitation-based Forster Resonance Energy Transfer (PIE-FRET) and Fluorescence Lifetime Imaging-based FRET (FLIM-FRET) to elucidate the composition of single nucleosomes containing Htz1p/H2A.Z in vitro and in vivo. We demonstrate yeast nucleosomes containing Htz1p are primarily comprised of H4 K12ac and H3 K4me3 but not H3 K36me3 and these patterns are conserved in mammalian cells. Quantification of epigenetic modifications in nucleosomes will provide a new dimension to epigenetics research and lead to a better understanding of how these patterns contribute to the targeting of chromatin-binding proteins and chromatin structure during gene regulation.