We aimed to describe bacterial etiology of acute otitis media (AOM) and characterize resistance, serotypes and genotype profiles of AOM-causing pneumococci recovered in Moscow children. Children with AOM and an available middle ear fluid specimen were prospectively enrolled in this study. Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis were considered as true otopathogens. All pneumococcal isolates were serotyped using the Quellung reaction; multidrug-resistant (MDR) pneumococci underwent multilocus sequence typing. In 172 of 541 enrolled AOM patients (32%) at least 1 otopathogen was recovered, with S. pneumoniae having the highest rate of 63% (109/172). When adjusted for antibiotic treatment before sampling, in untreated patients the rate of culture-positive AOM was 35% (124/352), S. pneumoniae had a prevalence of 69% (86/124), S. pyogenes 19% (24/124), H. influenzae 13% (16/124) and M. catarrhalis 9% (11/124). Among 107 examined pneumococci, 45% were penicillin-nonsusceptible, 34 and 30% were resistant to erythromycin and clindamycin, respectively; 30% had an MDR phenotype, but no amoxicillin-resistant isolates were found. Ten of 32 (31%) MDR pneumococci related to clonal complex 320, the remaining isolates belonged to 7 different clonal complex. Six leading serotypes were 19F (27%), 3 (12%), 6B (11%), 14 (11%), 19A (9%) and 23F (8%); overall polysaccharide conjugate vaccine13 coverage was 93%. S. pneumoniae, the leading bacterial AOM pathogen in Moscow children, is characterized by a substantial rate of antibiotic nonsusceptibility and clonality. A polysaccharide conjugate vaccine with expanded coverage seems to fit the current AOM pneumococcal serotype distribution in Russia better.