Penicillin-resistant strains of Streptococcus pneumoniae that are isolated with increasing frequency worldwide contain low-affinity penicillin-binding proteins (PBPs). The relatedness of PBPs from 55 resistant strains isolated on three continents was investigated by testing the reactivity of antibodies specific for PBP 1a or 2b and by comparing the PBP patterns. Seventeen patterns of antibody reactivity could be distinguished, 12 of which were specific to one isolate. Most strains, including all German and South African strains, had a unique PBP profile. A few groups of Spanish and Finnish isolates were identified where the strains within each group shared the same PBP profile, the same antigenic variants of PBPs 1a and 2b, and the same serogroup, suggesting that they represent different clones of S. pneumoniae. The results demonstrated highly variable pathways of resistance development and confirmed that resistant strains have emerged independently in different locations.