OBJECTIVE The objectives of this study were (i) to investigate the genetic background of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from Croatia and (ii) to monitor the prevalence of Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) among these isolates. METHODS Eighty-two hospital-acquired MRSA bloodstream isolates, collected in 2001 and 2002 in Croatia, were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). The presence of genes encoding PVL and TSST-1 was investigated by real-time PCR. RESULTS All strains were multiresistant and were distributed among 16 different similarity groups as determined by PFGE. Two of the groups, groups H and K, harboured the majority of the MRSA strains with 52 and 12%, respectively. The predominant SCCmec type found among the isolates was type I (89%). Eleven per cent of the strains harboured a modified SCCmec type III, which contained, in contrast to the regular type III, an additional dcs region. One strain harboured a novel SCCmec type, containing the ccrC gene in combination with the mecI gene, the dcs region, the locus between pI258 and Tn554 (locus E) and the locus between Tn554 and orfX (locus F). MLST showed the presence of ST111-MRSA-I and ST247-MRSA-I among Croatian MRSA isolates. All isolates were negative for both PVL and TSST-1. CONCLUSIONS These results indicate the emergence of ST111-MRSA-I and ST247-MRSA-I in Croatia among MRSA bloodstream isolates. The virulence factors PVL and TSST-1 were not present among these isolates.