Background: We conducted a cross-sectional study to examine the serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolated from nasopharynx of children attending daycare centers across South Korea, 4 years after the introduction of extended-valency pneumococcal conjugate vaccines ( PCVs). Methods: Between April and June 2014, a total of 734 nasopharyngeal swabs were collected from children aged 6-83 months attending 8 daycare centers located in 4 geographic areas across the country. Nasopharyngeal swabs were plated on trypticase soy agar with 5% sheep blood with gentamicn and incubated overnight at 37°C in 5% CO2. S. pneumoniae was identified based on the presence of alpha-hemolysis and inhibition by optochin. Serotype was determined by Quellung reaction using antisera. E-test was used to test the antibiotic susceptibility patterns (parenteral, nonmeningitis breakpoint for penicillin; ≤2.0ug/mL, susceptible; 4ug/mL, intermediate; ≥ 8ug/mL, resistant). Results: A total of 270 (36.4%) nasopharyngeal carriage isolates of pneumococci were identified, ranging between 30.1% and 57.5% across the daycare centers. The carriage rate was highest in children aged 12-23 months (51.3%), followed by 24-59 months (38.8%), ≥ 60 months (31.2%), and 6-11 months (25.0%). Of the 224 isolates after excluding non-typeable pneumococci, the most common serotypes were 23A (12.5%), followed by 15B (10.3%), 15C (19.4%), and 35B (7.1). The serotypes covered by PCVs accounted for 11.1%; while 88.9% were serotypes not covered by PCVs. Of the 75 isolates tested for antimicrobial susceptibility, 81.3% were susceptible, 14.7% were intermediate, and 4.0% were resistant to penicillin; 90.7% were susceptible, and 9.3% were intermediate to cefotaxime; and 6.7% were susceptible, 1.3% was intermediate, and 92.0% were resistant to erythromycin. Conclusion: This study demonstrated that carriage rate caused by any PCV serotypes decreased 4 years after the introduction of extended-valency PCVs in South Korea while carriage caused by non-PCV serotypes increased. Further study is needed to measure the vaccine impact on carriage.