Adenoviruses exhibit considerable intraserotypic genetic variability. Restriction enzyme analysis of the adenoviral genome is currently the most widely used procedure for the characterization of adenovirus isolates and has been extensively used for molecular epidemiological studies of subspecies B1 adenovirus infections. Comparison of restriction site maps between viral genomes is qualitatively consistent with DNA sequence homology providing that a sufficient number of sites are known. This technique is simple, sensitive, and can be adapted for screening numerous isolates and is therefore particularly useful for analysis of closely related genomes. Restriction enzyme analysis is still the only molecular approach that, at a reasonable cost, can give a “genome-wide” characterization of an adenovirus strain. Polymerase chain reaction (PCR) amplification followed by sequencing of the generated amplicon is the approach of choice for the detailed analysis of specific regions of the viral genome. Several laboratories have recently adopted PCR amplification of the hexon and/or fiber genes for the determination of adenovirus serotype identity, replacing identification by seroneutralization and hemmaglutination-inhibition. This approach permits rapid and objective type-specific identification of human adenoviruses and is especially useful for the characterization of serologically intermediate strains frequently identified among field strains of subspecies B1 adenoviruses.