Sepsis is a life-threatening disease with a high mortality rate. Rapid identification of blood culture isolates plays a crucial role in adequate antimicrobial therapy in sepsis patients. To accelerate microbiological diagnosis, a comprehensive panel of oligonucleotide probes for fluorescence in situ hybridisation (FISH) targeting Gram-positive cocci was compiled and evaluated on 428 positive blood culture specimens. By combining genus-specific and species-specific probes, the assay allowed discrimination of staphylococci, streptococci and enterococci as well as differentiation of therapy-relevant pathogens such as Staphylococcus aureus and Enterococcus faecium/durans. Furthermore, the newly designed FISH probes STREP2, ENCO and GRANU targeted Streptococcus pneumoniae/mitis, Enterococcus spp. (except E. faecalis) and Granulicatella adiacens group, respectively. The FISH assay achieved an overall sensitivity of 98.65% and a specificity of 99.0% and therefore allowed rapid and reliable molecular identification of Gram-positive cocci in blood culture specimens.