BACKGROUND: Pneumococcal conjugate vaccine (PCV) was introduced as a part of national immunization program to prevent invasive pneumococcal diseases in Indonesia in 2022. This study aims to investigate the serotype distribution, antimicrobial susceptibility, and genome profile of Streptococcus pneumoniae isolated from hospitalized patients from different regions in Indonesia prior to PCV introduction in Indonesia. MATERIALS AND METHODS: S. pneumoniae isolates were collected from blood, cerebrospinal fluid, and pleural fluid samples of hospitalised patients in three major referral hospitals located in Medan (North Sumatera), Semarang (Central Java), and Surabaya (East Java) from 2018-2021. Bacterial isolation and identification followed standard laboratory protocols. Serotyping was performed using conventional multiplex PCR and the Quellung reaction. Antimicrobial susceptibility testing (AST) was conducted by broth microdilution, and whole-genome sequencing (WGS) was carried out using the Illumina platform. RESULTS: S. pneumoniae isolates were recovered from 32 hospitalized patients. Serotype 19F (21.9%) was the most common followed by serotype 6B (12.5%) and 3 (9.4%). The serotypes of a high proportion of isolates (71.9%; 23/32) were included in the PCV13 vaccine. The majority of non-meningitis S. pneumoniae isolates were resistant to tetracycline (48.1%) followed by trimethoprim-sulfamethoxazole (33.3%), azithromycin and erythromycin (25.9% each), whereas penicillin resistant was found predominantly (60.0%) in meningitis isolates. We found AST and genomic prediction of antimicrobial resistance result was align in this study. We found 9 S. pneumoniae isolates (28.1%) were multidrug-resistant. Sequence type 1464 (n=5) and global pneumococcal sequence cluster 1 (n=7) was predominantly found in this study. CONCLUSION: Serotype 19F were the most common among vaccine-type serotype strains isolated from hospitalized patients prior to PCV introduction in Indonesia.