This work represents the first comprehensive quantitative analysis of global histone post-translational modifications (PTMs) from a virus infection, namely human cytomegalovirus (HCMV) infection. We used a nanoLC-MS/MS platform to identify and quantify the dynamic histone H3 and H4 PTMs expressed during HCMV replication in primary fibroblasts. Specifically, we examined the changes in histone PTMs over a 96 h time course to sample the immediate early (IE), early (E), and late (L) stages of viral infection. Several changes in histone H3 and H4 PTMs were observed, including a marked increase in H3K79me2 and H3K27me3K36me2, and a decrease in H4K16ac, highlighting likely epigenetic strategies of transcriptional activation and silencing during HCMV lytic infection. Heavy methyl-SILAC (hm-SILAC) was used to further confirm the histone methylation flux (especially for H3K79) during HCMV infection. We evaluated DOT1L (the H3K79 methyltransferase) mRNA levels in mock and HCMV-infected cells over a 96 h time course, and observed a significant increase in this methyltransferase as early as 24 hpi showing that viral infection up-regulates DOT1L expression, which drives H3K79me2. We then used shRNA to create a DOT1L knockdown cell population, and found that HCMV infection of the knockdown cells resulted in a 10-fold growth defect when compared with infected control cells not subjected to knockdown. This work documents multiple histone PTMs that occur in response to HCMV infection of fibroblasts, and provides a framework for evaluation of the role of epigenetic modifications in the virus-host interaction.