During spermiogenesis, nucleosome-based DNA packaging is replaced by protamine toroids to generate the highly compacted nucleus of the mature sperm of many species including human, mouse and bovine; however, the sperm from these species also retain some nucleosomes. It has been suggested that the histones of sperm nucleosomes may play important roles in embryo development. In the fruit-fly, Drosophila melanogaster it had been thought that nucleosomes and their component histones are completely replaced by protamine, and that no residual histones are retained. In this study, three experimental models, (Drosophila melanogaster, Mus musculus and Bos taurus) were used to investigate i) the presence of histone protein and/or its modifications in mature sperm, ii) paternal DNA packaging by histone, and iii) the possible epigenetic roles of sperm histones in embryogenesis. In this study, the presence of all the canonical histones of nucleosomal chromatin (H2A, H2B, H3 and H4) was confirmed in the mature sperm of Drosophila melanogaster by microscopic, biochemical and immunoprecipitation analysis. Evidence is also provided for the presence of post-translational modifications (acetylation and methylation) of the histones from fly, bovine and murine sperm. The molecular landscape of histones and histone modification (H3K27me3, H3K36me3 and H3K9me3) was investigated using ChiP-chip on a Drosophila-based, 2.4 million probe whole genome tiling array. Data analysis revealed that histones (and by extension, nucleosomes) are preferentially positioned within exons rather than introns and that they also mark intron/exon boundaries and gene promoters. However, nucleosomes are depleted upstream of Transcription Start Sites (TSS). Sperm chromatin also contains modified histones associated with gene regulation including repressive (H3K9me3, H3K27me3) and permissive (H3K36me3) marks. Gene ontology analysis showed that the H3K27me3 and H3K36me3 are associated with gene sequences carrying distinctive embryological developmental process terms. Moreover, the in silico analysis indicated that the early zygote expresses genes are marked by H3K36me3 in sperm chromatin, while the late zygote expresses genes that are marked by H3K9me3 instead. The same sequences are depleted of the H3K36me3 modification in sperm chromatin. These data suggest that modified sperm histones might play epigenetic roles in post fertilisation events such as formation and functioning of the male pronucleus and in early and late embryo development.