A novel TaqMan 5-plex real-time PCR using a combination of locked nucleic acid-modified (LNA)- and minor groove binding (MGB)-conjugated DNA probes was developed for identification and differentiation between the four main pathogenic Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. The assay also includes an exogenous internal control simultaneously verifying the PCR competency of the DNA samples. Validation of the novel assay was performed using DNA samples from 18 Brachyspira reference strains and 477 clinical samples obtained from porcine rectal swabs by comparing them with different PCR-based methods targeting nox, 16S rDNA, and 23S rDNA. The specificity of the assay was 100% without cross-reactivity or detection of different pathogens. Depending on the Brachyspira species, the limit of detection was between 10 and 20 genome equivalents with a cut-off threshold cycle (Ct) value of 37. The developed highly sensitive and specific 5-plex real-time PCR assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species recognized in pigs using a single-tube approach.