This study examined the prevalence of antibiotic-resistant clones that belong to serotypes not included in the pneumococcal conjugate vaccines and that cause a significant percentage of acute otitis media (AOM) in children in southern Israel. During 1998-2001, 2467 pneumococcal isolates, obtained from middle-ear fluid of children <3 years old with AOM, were characterized by antimicrobial susceptibility testing, serotype testing, and pulsed-field gel electrophoresis. Non-vaccine type (NVT) strains constituted 477 (19%) of the 2467 isolates, of which 173 (36%) belonged to only 4 serotypes: 35B, 33F, 21, and 15B/C. For serotype 35B, 47 (96%) of 49 strains were penicillin nonsusceptible, and 93% constituted a single clone; for serotype 33F, 31 (82%) of 38 strains were penicillin nonsusceptible, and 95% constituted a single clone; for serotype 21, 38 (93%) of 41 strains were penicillin nonsusceptible, and 93% constituted a single clone; for serotype 15B/C, 22 (49%) of 45 strains were penicillin nonsusceptible, and 42% constituted a single clone. Two of these clones have not been described elsewhere. The high prevalence of NVT clones should increase the awareness of the potential for replacement of the vaccine strains with these NVT antibiotic-resistant strains.