Abstract Background Enterovirus 68 (EV68) belongs to species Human enterovirus D. It is unique among enteroviruses because it shares properties with human rhinoviruses. After the first isolation in 1962 from four children with respiratory illness, reports of (clusters of) EV68 infections have been rare. During the autumn of 2010, we noticed an upsurge of EV68 infections in the Northern part of the Netherlands in patients with severe respiratory illness. Objectives To give a detailed description of the clinical and virological data of patients with EV68 infection identified in 2010, and compare these with data collected in 2009. Study design We systematically collected clinical data from patients with an EV68 infection detected in 2010. We added four patients with an EV68 infection from 2009. Further characterization of EV68 was performed by partial sequence analysis of the VP1 genomic region. Results In 2010, EV68 was identified as the only cause of respiratory illness in 24 patients, of which 5 had to be admitted to the intensive care unit. Sequence analysis revealed different lineages in the majority of EV68 detected in 2010 as compared to the 2009 isolates. Conclusions We noticed an increase of EV68 infections and present clinical as well as sequence data, in which two distinct phylogenetic clusters could be identified.