Comparative genomic and computational analyses of four genomes of human adenovirus type 4 (HAdV-E4), spanning fifty years, document the molecular evolution of this pathogen at the nucleotide level by revealing a recent recombination with HAdV-B within the DNA replication signals in the Inverted Terminal Repeat of two recently circulating isolates. This, along with an earlier recombination found within the prototype genome that includes the hexon gene of HAdV-B16, apparently allows the current, most virulent isolates to be established in the population.