Toxin-antitoxin (TA) systems consist of toxin-inhibiting diverse cellular functions (e.g., DNA replication, transcription, and translation) and a noncoding RNA or protein antitoxin. TA systems are associated with various cellular events, such as stress responses, programmed cell death, and bacterial pathogenicity. Recent advances in genome sequencing and bioinformatics research have demonstrated that most bacteria harbor various kinds of TA modules on their chromosomes; however, there is little understanding of chromosomally encoded TA systems in the Gram-positive pathogen Here, we report on newly discovered TA systems, each of which is composed of two proteins. Manual search and gene operon prediction analysis identified eight 2-gene operons as potential candidates for TA systems. Subsequently, using an host killing and rescue assay, we demonstrated that four of the eight candidates worked as TA systems, designated , , , and Moreover, the TsaT, TsbT, TscT, and TsdT toxins inhibited growth, and the toxicity of TsbT was neutralized by coexpressing the gene in the native host, Further, the bioinformatics analysis of the gene clusters revealed that TsaAT, TsbAT, TscAT, and TsdAT did not exhibit sequence similarity to known bacterial TA systems, and their homologues were present only within species and not among any other bacteria. Our results further advance not only the understanding of TA systems but also the study of unannotated TA systems in various bacterial species. Recent advances in genome sequencing and bioinformatics research have demonstrated that most pathogenic bacteria harbor a large number of chromosomally encoded toxin-antitoxin (TA) modules. However, little is known about the TA systems in Here, we newly identified four TA systems using a combination of manual base-by-base screening and functional analysis in Moreover, all toxins of the identified TA systems caused growth inhibition in the native host Although the newly identified TA systems did not exhibit sequence similarity with known bacterial TA systems, their orthologues were conserved only among other species, indicating their uniqueness to staphylococci. Our approach opens the possibility for studying unannotated TA systems in various bacterial species.