Around two thirds of sudden unexpected deaths in infancy (SUDI) remain unexplained following post-mortem examination. It has been postulated that a subset of unexplained SUDI may be caused by toxigenic Staphylococcus aureus. The aim of this study was to compare the prevalence of toxigenic S aureus strains in unexplained and explained SUDI (those in whom a cause of death is determined at autopsy). A retrospective review was performed of 546 SUDI autopsies as part of a larger review of >1,500 pediatric autopsies over a 10-year period, 1996-2005 inclusive. SUDI was defined as the sudden and unexpected death of an infant aged 7-365 days, and categorized into unexplained, explained with histological evidence of infection (bacterial infection group) or explained due to non-infective causes. Toxin gene profiling was carried out by PCR in cases in whom S aureus was isolated as part of clinical investigation. Of the 507 SUDI included in this analysis, bacteriological investigations were performed in 470, and S aureus was isolated on post-mortem culture from at least one site in 173 (37%). There were significantly more cases with S aureus isolated in unexplained SUDI (40%) compared to non-infective SUDI (21%; difference 19.0%, 95% CI 5.4% to 29.3%, P = 0.006). 46% of all cases with S aureus isolated underwent routine testing for a panel of staphylococcal toxin genes (including SEA to SEE, SEG to SEJ, TSST-1, and exfoliative toxins A and B). There were more cases with at least one toxigenic strain of S aureus in the unexplained SUDI (81%) and bacterial infection groups (77%) than in the non-infection group (63%), but these differences were not statistically significant (Fisher exact test, P = 0.44). Toxin gene-carrying S aureus is commonly detected at autopsy in SUDI, accounting for 78% of S aureus isolates submitted for toxin gene profiling in this series. There is a significantly higher prevalence of S aureus in unexplained SUDI compared to non-infective SUDI, but no significant difference in the proportion with toxigenic S aureus strains isolated between the groups. These data are consistent with the hypothesis that a subset of otherwise unexplained SUDI may be related to the presence of S aureus colonization/infection, but do not indicate routine testing for toxin-associated genotypes.