With the introduction of the Haemophilus influenzae serotype b (Hib) vaccine, invasive Hib disease has decreased substantially, but nontypeable H. influenzae (NT Hi) disease appears to be increasing. In order to understand the origin of NT Hi strains and their relationship with serotypeable strains, we analysed 125 NT Hi isolates collected from individual patients with either invasive disease (70 isolates) or respiratory tract infections (55 isolates). Serotype-specific and capsular transport genes were absent by PCR analysis, confirming their nonencapsulated status, which also suggested the NT Hi isolates were not encapsulated strains that shed their capsules. Multilocus sequence typing confirmed the NT Hi isolates did not have the same genetic background as serotypeable strains, including Hib. Despite the genetic heterogeneity found, two major genetic clusters were identified, both containing invasive and respiratory isolates. Fourteen invasive isolates and nine respiratory isolates produced beta-lactamase and were ampicillin resistant. More invasive (26.8%) than respiratory isolates (10.9%) showed decreased susceptibility towards ampicillin by a mechanism unrelated to beta-lactamase production. Besides a change in the capsule status of invasive Hi strains, the burden of invasive Hi disease, which used to be mainly a childhood disease, has now shifted to involve both adults and infants.