BackgroundStaphylococcus aureus(S. aureus) infection-induced osteomyelitis (OM) is an inflammatory bone disease accompanied by persistent bone destruction, and the treatment is challenging because of its tendency to recur. Present study was aimed to explore the molecular subgroups ofS. aureusinfection-induced OM and to deepen the mechanistic understanding for molecularly targeted treatment of OM.MethodsIntegration of 164 OM samples and 60 healthy samples from three datasets of the Gene Expression Omnibus (GEO) database. OM patients were classified into different molecular subgroups based on unsupervised algorithms and correlations of clinical characteristics between subgroups were analyzed. Next, The CIBERSORT algorithm was used to evaluate the proportion of immune cell infiltration in different OM subgroups. Weighted gene co-expression analysis (WGCNA) was used to identify different gene modules and explore the relationship with clinical characteristics, and further annotated OM subgroups and gene modules by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.ResultsTwo subgroups with excellent consistency were identified in this study, subgroup and hospital length of stay were independent predictors of OM. Compared with subgroup I, OM patients in subgroup II had longer hospital length of stay and more severe disease. Meanwhile, the infiltration proportions of monocytes and macrophages M0 were higher in patients of OM subgroup II. Finally, combined with the characteristics of the KEGG enrichment modules, the expression of osteoclast differentiation-related genes such as CTSK was upregulated in OM subgroup II, which may be closely associated with more severe OM patients.ConclusionThe current study showed that OM subgroup II had longer hospital length of stay and more severe disease, the osteoclast differentiation pathway and the main target CTSK contribute to our deeper understanding for the molecular mechanisms associated withS. aureusinfection-induced OM, and the construction of molecular subgroups suggested the necessity for different subgroups of patients to receive individualized treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12920-023-01568-x.