Abstract Background The objective of this study was to determine the prevalence of nasopharyngeal carriage, serotype distribution, and penicillin resistance of Streptococcus pneumoniae in children ≤6 years of age in Ghana. Methods A cross-sectional study was carried out on a cluster-randomized sample of children ≤6 years of age attending nurseries and kindergartens in Accra and Tamale, Ghana. Basic data on age, sex and exposure to antimicrobials in the previous month were collected on all study subjects. Nasopharyngeal swabs were obtained from participants and all pneumococcal isolates were characterized by serotyping and their penicillin resistance determined. Results The overall prevalence of pneumococcal carriage among the children was 34% in Accra and 31% in Tamale. The predominant serotypes were 19F, 6B, 23F, and 6A with 23% of the isolates being non-typable in Accra and 12% in Tamale. Only two isolates (serotypes 19F and 6B) from Tamale had a MIC >2 μg/ml and were classified as fully penicillin resistant with 45% of the isolates having intermediate resistance. Conclusions These findings indicate that the 13-valent pneumococcal conjugate vaccine (PCV-13) recently introduced in Ghana will cover 48% and 51% of the serotypes identified in Accra and Tamale, respectively. The 23-valent pneumococcal polysaccharide vaccine (PPV-23) will cover 54% of all serotypes detected. The two penicillin resistant isolates (MIC 32 μg/ml) were serotypes included in both PCV-13 and PPV-23. A nationwide monitoring system of penicillin susceptibility patterns and pneumococcal serotypes is recommended.