Streptococcus pneumoniae is a leading cause of respiratory and invasive infections, particularly in children under five and immunocompromised adults. Pakistan introduced the PCV-10 vaccine in 2012 to reduce pneumococcal disease burden. This study investigates post-vaccine serotype replacement and the emergence of non-vaccine strains in Pakistan. Clinical and carriage isolates were analyzed through culture, optochin susceptibility, and multiplex PCR for lytA and pspA genes to confirm serotypes. Findings indicate a marked decline in vaccine-covered serotypes but a growing prevalence of non-vaccine and vaccine-escape strains, especially in pus, sputum, and blood samples. The geographic variation of serotypes highlights the need for region-specific vaccination strategies and continuous surveillance to maintain vaccine effectiveness.