This study reports the identification of a new human coronavirus, HCoV-NL63, using a novel virus discovery method called Virus-Discovery-cDNA-AFLP (VIDISCA). The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence revealed that HCoV-NL63 is a new group 1 coronavirus, distinct from known human coronaviruses HCoV-229E and HCoV-OC43. HCoV-NL63 replicates efficiently in monkey kidney cells and has a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals with respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating widespread circulation within the human population. The study highlights the importance of continuous search for new viruses to understand the etiology of various human diseases.This study reports the identification of a new human coronavirus, HCoV-NL63, using a novel virus discovery method called Virus-Discovery-cDNA-AFLP (VIDISCA). The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence revealed that HCoV-NL63 is a new group 1 coronavirus, distinct from known human coronaviruses HCoV-229E and HCoV-OC43. HCoV-NL63 replicates efficiently in monkey kidney cells and has a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals with respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating widespread circulation within the human population. The study highlights the importance of continuous search for new viruses to understand the etiology of various human diseases.