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ABSTRACTWe used pulsed-field gel electrophoresis to study the epidemiology and population structure of Haemophilus influenzaetype b. DNAs from 187 isolates recovered between 1985 and 1993 from Aboriginal children (n= 76), non-Aboriginal children (n= 106), and non-Aboriginal adults (n= 5) in urban and rural regions across Australia were digested with the SmaI restriction endonuclease. Patterns of 13 to 17 well-resolved fragments (size range, ~8 to 500 kb) defining 67 restriction fragment length polymorphism (RFLP) types were found. Two types predominated. One type (n= 37) accounted for 35 (46%) of the isolates from Aboriginals and 2 (2%) of the isolates from non-Aboriginals, and the other type (n= 41) accounted for 2 (3%) of the isolates from Aboriginals and 39 (35%) of the isolates from non-Aboriginals. Clustering revealed seven groups at a genetic distance of ~50% similarity in a tree-like dendrogram. They included two highly divergent groups representing 50 (66%) isolates from Aboriginals and 6 (5%) isolates from non-Aboriginals and another genetically distinct group representing 7 (9%) isolates from Aboriginals and 81 (73%) isolates from non-Aboriginals. The results showed a heterogeneous clonal population structure, with the isolates of two types accounting for 42% of the sample. There was no association between RFLP type and the diagnosis of meningitis or epiglottitis, age, sex, date of collection, or geographic location, but there was a strong association between the origin of isolates from Aboriginal children and RFLP type F2a and the origin of isolates from non-Aboriginal children and RFLP type A8b. The methodology discriminated well among the isolates (D= 0.91) and will be useful for the monitoring of postvaccine isolates of H. influenzaetype b.