Subgenus identification of adenoviruses is of clinical importance and is as informative as identification by serotype in most clinical situations. A PCR-based identification of adenovirus subgenera A, B, C, D, E, and F and sometimes serotypes is described. The PCR uses nonnested primer pair ADRJC1-ADRJC2, which targets a highly conserved region of the adenovirus hexon gene, has a sensitivity of 10 to 40 copies of adenovirus type 2 (Ad2) DNA, and generates 140-bp PCR products from adenovirus serotypes representative of all the subgroups. The PCR products of all subgroups can be differentiated on the basis of the restriction fragment patterns produced by a total of five restriction endonucleases. In addition, serotypes Ad40 and Ad41 (subgroup F) and important serotypes of subgroup D (Ad8, Ad10, Ad19, and Ad37) can easily be differentiated, but serotypes within subgroups B and C cannot. The method was assessed by blind subgenus identification of 56 miscellaneous clinical isolates of adenoviruses. The identities of these isolates at the subgenus level by the PCR correlated 91% (51 of 56) with the results of serotyping by the neutralization test, and 9% (5 of 56) of clinical isolates produced discordant results.