Infections by nonpolio enteroviruses (EVs) are highly prevalent, particularly among children and neonates, where they may cause substantial morbidity and mortality. Laboratory diagnosis of these viral infections is important in patient prognosis and guidance of clinical management. Although the laboratory diagnosis of non-polio EVs is mainly based on molecular techniques, classical virus-isolation techniques are still used in reference laboratories. Other techniques, such as antigen detection and serology, are becoming obsolete and rarely used in diagnosis. An important part of diagnosis and surveillance of EV infections is viral typing by VP1 gene sequencing using conventional Sanger technique and more recently, full-genome next-generation sequencing. The latter allows the typing of all EVs, better investigation of EV outbreaks, detection of coinfection, and identification of severity markers in the EV genome.