Monitoring antibiotic-resistant pneumococcal strains not covered by the 7-valent conjugate vaccine is an important priority. The Centers for Disease Control and Prevention's Active Bacterial Core Surveillance identified 68 invasive penicillin-nonsusceptible serotype 35B (PN35B) isolates recovered from 1995 to 2001 from patients residing in the states of California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New York, Oregon, Tennessee, and Texas. Nonsusceptible isolates accounted for 69% of all serotype 35B isolates recovered during this time. Twelve (18%) of the 68 PN35B isolates recovered since 1995 were obtained from pediatric patients. These 68 isolates exhibited penicillin MICs of 0.25-2 microg/mL and reduced susceptibility to cefotaxime. Representative PN35B isolates exhibited a common chromosomal macrorestriction profile and identical penicillin-binding-protein gene restriction profiles characteristic of penicillin-resistant strains, and they shared a unique 7-locus sequence type that included 3 new alleles. The mosaic pbp2b and divergent ddl sequences were suggestive of interspecies recombination at the ddl-pbp2b chromosomal region.