Optochin susceptibility testing is a major assay used for presumptive identification of Streptococcus pneumoniae. Still, atypical optochin-resistant (Opt) pneumococci have been reported and this phenotype has been attributed to nucleotide substitutions in the genes coding for the FFATPase. While substitutions in the atpC gene (c-subunit of ATPase) are more common and better characterized, data on mutations in the atpA (a-subunit) are still limited. We have characterized five Opt isolates presenting alterations in the atpA (Trp206Cys in four isolates and Trp206Ser in one isolate), constituting the first report of such mutations in Brazil. Most of the Opt isolates consisted of heterogeneous populations. Except for Opt MICs and the nucleotide changes in the atpA gene, Opt and Opt subpopulations originating from the same culture had identical characteristics. In addition, we compared phenotypic and genetic characteristics of these atpA mutants with those of atpC mutants previously identified in Brazil. No structural alterations were detected among predicted proteins, regardless of mutations in the coding gene, suggesting that, despite the occurrence of mutations, protein structures tend to be highly conserved, ensuring their functionalities. Phylogenetic analysis revealed that atypical Opt strains are true pneumococci and Opt resistance does not represent any apparent selective advantage for clinical isolates.