The precise mechanisms that contribute to the long-term persistence of otitis media (OM) in patients remain incompletely comprehended. Comprehending the molecular mechanisms that assist bacteria in persisting within the middle ear during otitis media (OM) could unveil the essential pathways necessary for the condition. The objective of this study is to isolate and identify Streptococcus pneumoniae from patients with otitis media in Hilla governorate. In this study, certain individuals were identified using standard phenotypic approaches, while others were identified using both phenotypic and genotypic methods, specifically the monoplex-PCR methodology. All isolates showed positive amplification when specific primers for S. pneumoniae were used to detect the lytA and ply genes at the molecular level. Conclusion: Out of 100 clinical samples, analysis of the virulence gene showed that all samples of Streptococcus pneumoniae contained both the lytA and ply gene. The genes provide the genetic instructions for producing the capsule , recognized for its exceptional effectiveness in protein analysis and tissue death .