The aim of this study was to characterize invasive penicillin non-susceptible pneumococci (PNSP) isolated in Denmark from 2001 to 2005, describe the genetic changes in the penicillin-binding proteins (PBPs) and compare the isolates to the international recognized PNSP clones. All isolates were characterized using serotyping; 43 isolates were also characterized by multilocus sequence typing (MLST) and sequence determination of the PBP genes, pbp1a, pbp2b and pbp2x. In addition, isolates were tested for susceptibility to various antimicrobial agents. From 2001 to 2005, 178 invasive PNSP isolates were found among all invasive isolates. Of these, 13% were penicillin-resistant pneumococci (PRP). The PRP isolates were different to the penicillin intermediate-resistant pneumococci (PIRP) as they were co-resistant to a higher number of antimicrobial agents, and the serotype diversity was lower for the PRP isolates compared to the PIRP isolates. Changes in the PBPs were found to be the same as those observed in other studies. Most PRP isolates were found to belong to the clonal complex CC156 of which most were ST156 serotype 9V or 14. ST156 is a well known multiresistant widespread international PMEN (Pneumococcal Molecular Epidemiology Network) clone, Spain9V-3, which is also present as one of the dominant clones in our neighbour countries of Sweden, Norway and Germany.