Species Human mastadenovirus E (HAdV-E) comprises several simian types and a single human type: HAdV-E4, a respiratory and ocular pathogen. RFLP analysis for the characterization of intratypic genetic variability has previously distinguished two HAdV-E4 clusters: prototype (p)-like and a-like. Our analysis of whole genome sequences confirmed two distinct lineages, which we refer to as phylogroups (PGs). PGs I and II comprise the p- and a-like genomes, respectively, and differ significantly in their G + C content (57.7% ± 0.013 vs 56.3% ± 0.015). Sequence differences distinguishing the two clades map to several regions of the genome including E3 and ITR. Bayesian analyses showed that the two phylogroups diverged approximately 602 years before the present. A relatively faster evolutionary rate was identified for PG II. Our data provide a rationale for the incorporation of phylogroup identity to HAdV-E4 strain designation to reflect the identified unique genetic characteristics that distinguish PGs I and II.