Background: Alpha-hemolysin (Hla) is a major virulence factor in the pathogenesis of Staphylococcus aureus infection, being active against a wide range of host cells. Although hla is ubiquitous in S. aureus, its genetic diversity and variation in expression in different genetic backgrounds is not known. We evaluated nucleotide sequence variation and gene expression profiles of hla among representatives of hospital (HA) and community-associated (CA) S. aureus clones. Methods: 51 methicillin-resistant S. aureus and 22 methicillin-susceptible S. aureus were characterized by PFGE, spa typing, MLST and SCCmec typing. The internal regions of hla and the hla promoter were sequenced and gene expression was assessed by RT-PCR. Results: Alpha-hemolysin encoding- and promoter sequences were diverse, with 12 and 23 different alleles, respectively. Based on phylogenetic analysis, we suggest that hla may have evolved together with the S. aureus genetic background, except for ST22, ST121, ST59 and ST93. Conversely, the promoter region showed lack of co-evolution with the genetic backgrounds. Four non-synonymous amino acid changes were identified close to important regions of hla activity. Amino acid changes in the RNAIII binding site were not associated to hla expression. Although expression rates of hla were in general strain-specific, we observed CA clones showed significantly higher hla expression (p = 0.003) when compared with HA clones. Conclusion: We propose that the hla gene has evolved together with the genetic background. Overall, CA genetic backgrounds showed higher levels of hla expression than HA, and a high strain-to-strain variation of gene expression was detected in closely related strains.