Culture and serotyping of human enteroviruses are time-consuming and labor-intensive. Targeted nucleic acid sequencing has emerged as a powerful alternative to conventional methods. Many published genotyping assays use two-step reverse transcription and polymerase chain reaction (RT-PCR), nested PCR protocols, and/or reflexive testing algorithms. The performance of a one-step RT-PCR protocol, a more clinically practical approach, was evaluated. The VP1 and/or VP2 region of archived enterovirus isolates (n=36, representing 32 serotypes), patient enterovirus isolates not typeable by immunofluorescence antibodies (n=50), and enterovirus from direct patient specimens (48 cerebrospinal fluid, 2 plasma/serum, 1 blood) were amplified and sequenced for genotype identification. The analytical sensitivity of the genotyping assays was 100-fold less than detection by RT-PCR of the 5\'-untranslated region. Thirty-four of 36 archived isolates could be genotyped by combining results of VP1 and VP2 target sequencing. Non-typeable isolates included 17 echovirus 18, 6 enterovirus 68, 6 rhinovirus, and 7 which could not be classified further. From clinical specimens, 23 of 51 (45%) could be identified using VP2 typing and the most common types were coxsackievirus B1, echovirus 30, and echovirus 6. Using a one-step RT-PCR without nesting, most enterovirus isolates and a subset of clinical samples with high viral titer could be genotyped.