A recent study of pneumococcal colonization in 3198 healthy children of 1-19 years of age in The Netherlands showed pneumococcal colonization in 19 % of the children, with a peak incidence of 55 % at the age of 2 years; an age-related serotype distribution was also found. In the present study, the genetic background and resistance profiles of 578 pneumococcal isolates from the latter study were characterized by means of chromosomal genotyping and susceptibility testing. In contrast to the age-related serotype distribution observed previously, the genetic background of the strains was not age related. Few strains were found showing close homology (>95 %) with the international clones Spain(9V)-3 (ten isolates showed homology), England(14)-9 (four isolates), Tennessee(23F)-4 (two isolates), CSR(14)-10 (one isolate) and Sweden(15A)-25 (one isolate). In total, 19 % of strains showed resistance to one or more antibiotics. Resistance to cotrimoxazole, tetracycline, erythromycin and penicillin was found in 12.9, 5.6, 5.0 and 2.7 % of isolates, respectively. Multidrug resistance was found in 1.9 % of strains. In conclusion, pneumococcal colonization isolates from healthy Dutch children represent a heterogeneous, mostly antibiotic susceptible, genetic population.