The impact of the 7-valent pneumococcal conjugate vaccine (PCV7 [Prevnar]) on penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) recovered from children with acute otitis media (AOM) is unclear. At 5 hospitals, 505 pneumococcal isolates were collected from children with AOM between 1 January 1999 and 31 December 2002. Molecular subtyping was performed on 158 isolates. Overall, the percentage of AOM cases due to non-PCV7 serogroups (including serotype 3) increased over time (from 12% in 1999 to 32% in 2002; P < .01) and according to the number of PCV7 doses received (18% [< or = 1 dose] vs. 35% [2-4 doses]; P < .01). The percentage of cases due to vaccine-related serotypes (including serotype 19A) increased according to the number of PCV7 doses received (10% [< or = 1 dose] vs. 19% [2-4 doses]; P = .05) but not over time, whereas the percentage of cases due to serotype 19F remained unchanged both over time and according to the number of PCV7 doses received. The frequency of penicillin nonsusceptibility among PCV7 serotypes (range, 65%-75%) and non-PCV7 serogroups (range, 11%-27%) did not significantly change overall. Although no change was detected among isolates collected from children with spontaneous drainage, the percentage of pneumococci recovered at the time of myringotomy and/or tympanostomy tube placement that were nonresistant to penicillin decreased over time (from 73% in 1999 to 53% in 2002; P = .03). All of the serotype 3 strains were genetically related, whereas 88% of the isolates that were either serotype 19F or serotype 23F were related to 1 of 3 international clones. Among children with AOM, the proportion of cases due to non-PCV7 serogroups increased, vaccine-related serotypes increased, and serotype 19F remained unchanged. Although a decrease in the proportion of cases due to PNSP occurred among children who required myringotomy and/or tympanostomy tube placement, the proportion of PNSP remained unchanged overall and among children with spontaneous drainage. Because future trends in the susceptibility patterns of pneumococcal isolates recovered from children with AOM are not easy to predict, continued surveillance is essential.