A new reverse transcription-polymerase chain reaction assay was developed for identification of 28 Canadian human parechovirus (HPeV) isolates, including 20 HPeV-1, 3 HPeV-2, and 5 HPeV-3, recovered from 1985 to 2004. All HPeV-1 isolates but 1 were genetically distinct from the Harris reference strain. One HPeV-2 isolate was related to the Williamson strain; the other 2 were related to the Connecticut strain. HPeV-3 isolates clustered together. Seventy-five percent of isolates were recovered during the typical enterovirus season. All patients but 1 were children with a mean age of 14.6 months, 6.3 months, and 0.7 months for HPeV-1, HPeV-2, and HPeV-3 patients, respectively. All HPeV-2- and HPeV-3-infected children were hospitalized with a diagnosis of viremia or sepsis. HPeV-1- infected children had bronchiolitis diagnosed in 50% of the cases, with few cases of pneumonia and enteritis. Two infected patients (1 child with leukemia and a 78-year-old woman) died of septic shock and severe pneumonia, respectively.