One of the beneficial effects of pneumococcal conjugate vaccines (PCVs) has been a decrease in the incidence of non-invasive infections, such as otitis media (OM) caused by vaccine serotypes. In this study, we analyzed the epidemiology of pneumococcal OM before and after PCV13 introduction in 2010. Between 2008 and 2016, the middle ear exudates from 2653 children under 14 years of age with OM were studied in two Spanish provinces (Gipuzkoa and Barcelona), and S. pneumoniae was isolated in 235 (8.9%) of cases. The 204 available isolates were serotyped and distributed in three 3-year periods: one before and two after PCV13 introduction (early and late post-PCV13). A significant decrease in the rate of OM caused by S. pneumoniae was observed mainly due to a decrease in infections caused by all PCV13 serotypes, although exceptions were observed including the persistence of serotype 3 in Gipuzkoa and a weak re-emergence of serotype 19F in both regions. The rate and diversity of non-PCV13 serotypes increased in both regions and an emerging clone causing OM was detected in each region: serotype 23B ST2372 in Gipuzkoa and serotype 11A ST838/ST6521 in Barcelona. The introduction of PCV13 has been followed by a change in the epidemiology of pneumococcal OM, with a decrease in the rate of vaccine serotypes accompanied by an increase in the diversity of non-vaccine serotype and the clonal spreading of different single clones in each region.