Objectives:Recently, a new lineage of MRSA with Sequence Type 398 (ST398) has emerged throughout the world. This lineage has been isolated from patients with invasive community-associated MRSA infections and colonization rates among contacts of livestock are very high, although the strains until now have mainly been associated with colonization and infection of livestock, such as pigs and calves. By determining the genome sequence of isolate S0385 a ST398 MRSA carrying SCCmec type V we have identified several genetic elements that may explain the success of this lineage. However the presence of at least two different SCCmec elements among MRSA ST398 strains might indicate significant differences in genomic content between strains. To obtain better insight in the differences and clonality of S. aureus ST398 lineage, the complete genome sequences of 3 additional isolates all carrying SCCmec type IV were determined. Methods: Isolates S0452, S0460 and S0462 were isolated on the same farm, but from different clinical backgrounds. The sequence of the genomes was determined using either 454 or Solexa based sequencing technology. Resulting contigs were aligned against the genome of isolate S0385 for a putative direction and order. Predicted ORFs were matched against the 10 complete genomes currently published and if no homology was found a search in the non-redundant nucleotide sequence database was performed. DNA comparisons were performed using Kodon. Results: Comparison of four S. aureus ST398 isolates showed considerable differences between S0385 and the other three isolates. Over 200 single nucleotide polymorphisms (SNPs) were found in the core chromosome with the majority of SNPs located in coding regions. The difference in gene content between the ST398 isolates was almost exclusively restricted to genes related to mobile genetic elements (MGE). Besides the difference in SCCmec elements major differences in phage, integrative conjugative element or plasmid gene content were observed between isolates. On the other hand several MGE were present in all ST398 isolates, such as a unique pathogenicity island encoding two excreted virulence factors and transposons conferring tetracycline and β-lactamase resistance. In addition, novel allelic variants of the νSa islands containing only a single type 1 restriction and modification system and lacking several virulence factors like enterotoxins were conserved among all isolates. Conclusions: MRSA ST398 strains have undergone substantial genetic diversification, in particular with respect to the acquisition of mobile genetic elements. These findings suggest MRSA ST398 has diverged independently from a common ancestor on at least two occasions.