Background: Streptococcus pneumoniae causes significant morbidity and mortality worldwide. Identification of S. pneumoniae colonization in the nasopharynx is critical for understanding the pathogenesis of pneumococcal diseases and for estimation of vaccine efficacy.Aim of the work: We aimed to detect the rate of nasopharyngeal carriage of S. pneumoniae among children less than 5 years and to use molecular methodology for the detection.Methods: We investigated the colonization characteristics for these pathogens among 200 non- infected children from 6 months to 5 years of age in Cairo, Egypt, in the winter season 2012/2013. Nasopharyngeal specimens were collected using flocked swabs. The specimens were cultures and tested by PCR for detection of Lyt-A and Ply genes. Antimicrobial susceptibility was done using the disk diffusion method and the E test method.Results: The prevalence of S. pneumoniae colonization was 31% as detected by culture and 56.5% by PCR targeting lytA and ply genes when CT 35 was the cutoff value. We observed a high prevalence of pneumococcal resistance against trimethoprim–sulfamethoxazole (96.7%), tetracycline (72.6%), penicillin (72.6%), erythromycin (50%), and clindamycin (35.5%). The MIC for penicillin was 0.75ug/ ml or less for 58 out of 62 isolates (93.5%), 1.5 ug/ml for 2 isolates (3.2%), and 4ug/ml for 2 isolates (3.2%). Conclusion: The pneumococcal NP carriage rate is high among children in our community with increased antimicrobial resistance rates. The PCR provides a rapid and highly sensitive molecular method in detection of pneumococcal carriage. Our data form an important basis to monitor the future impact of pneumococcal vaccination on bacterial colonization, as well as to recommend a rationalized and restrictive antimicrobial use in our community.