Amplified fragment length polymorphism versus pulsed-field gel electrophoresis was used for fingerprinting of 85 macrolide-resistant pneumococcal isolates identified by using primarily phenotypic methods. Confirmation of identification by 16S rRNA sequencing revealed that 27 isolates were actually nonpneumococci. Amplified fragment length polymorphism but not pulsed-field gel electrophoresis offered simultaneous and accurate discrimination between pneumococci and nonpneumococcal species.