Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants.