The phylogenetic relationships of non-typable Haemophilus influenzae (NTHi) strains prospectively isolated from healthy children and children with acute otitis media (AOM) were analysed using multilocus sequence typing (MLST). A total of 165 NTHi isolates were collected over a 3.5 year time frame during 2006 through 2009. The strains were tested for β-lactamase production; 28.5 % were positive. Seventy different NTHi sequence types (STs) were identified of which 29 (41.4 %) were novel. NTHi strains did not show any phylogenetic grouping or clustering among asymptomatic colonizing strains or strains that caused AOM, or based on β-lactamase enzyme production. Evaluation of triplets and other siblings over time demonstrated relatively frequent genetic exchanges in NTHi isolates in vivo in a short time frame and subsequent transfer among children in a family. Comparison of the MLST STs isolated at different time points showed that in ∼85 % of the nasopharynx (NP) colonizations, NTHi strains cleared from the host within 3 months, that sequential colonization in the same child involved different strains in all cases except one, and that NP and middle ear isolates were identical STs in 84 % of cases. In this first study of its type to our knowledge, we could not identify predominant MLST types among strains colonizing the NP versus those causing AOM or expressing a β-lactamase enzyme conferring penicillin resistance in children.