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• Among 141 Streptococcus pneumoniae strains, 63.8% and 69.5% were resistant to penicillin G and erythromycin, respectively. • Ninety-three isolates (66.0%) were multidrug-resistant. • The serotypes detected were covered by PVC7, PCV10 and PCV13 in 56.7%, 60.3% and 75.2% of isolates, respectively. • Among the 73 sequence types (STs) identified, 23 were described for the first time. • Clonal dissemination of 3 international resistant clones, including Denmark14-ST230, Spain9V-ST156 and Portugal19F-ST177. ABSTRACT Objectives This study aimed to explore the genetic diversity of Streptococcus pneumoniae isolates in a Tunisian pneumology hospital. Methods A total of 141 S. pneumoniae strains isolated between 2009–2016 in the microbiology laboratory at A. Mami Hospital of Pneumology were investigated. Antimicrobial susceptibility testing was performed the disk diffusion method. MICs of penicillin G, amoxicillin and cefotaxime were determined by Etest. Serotyping was inferred from the results of multiplex PCR targeting 40 serotypes. Sequence types (STs) were determined by multilocus sequence typing (MLST). Results Among the 141 S. pneumoniae isolates, 98 (69.5%) were resistant to erythromycin. Evaluation of β-lactam susceptibility showed that 90 strains (63.8%) were non-susceptible to penicillin, whereas 48 (34.0%) had decreased susceptibility to amoxicillin and 21 (14.9%) to cefotaxime. Twenty-five serotypes were detected, and 10 isolates were classified as non-typeable. Vaccine coverage was 56.7%, 60.3% and 75.2% for pneumococcal conjugate vaccine 7 (PCV7), PCV10 and PCV13, respectively. Overall, 73 STs were identified, including 23 described for the first time. The most frequent STs were ST179 (n = 17), ST3772 (n = 14), ST2918 (n = 10) and ST4003 (n = 5), related to serotypes 19F, 19A, 14 and 23F, respectively. Moreover, 110 strains were classified within 45 STs. Three international antimicrobial-resistant clones were found, including Denmark14-ST230 (n = 22), Spain9V-ST156 (n = 22) and Portugal19F-ST177 (n = 20). Conclusion This study emphasises the clonal and international dissemination of antimicrobial-resistant S. pneumoniae clones. Significant differences in genetic variation were documented by MLST within the various serotypes identified. [ABSTRACT FROM AUTHOR]