Streptococcus (S.) dysgalactiae subsp. equisimilis is responsible for severe diseases in humans, including primary bacteraemia, pneumonia, endocarditis, and toxic shock syndrome. Infection in some animal species can also occur, although a few studies have looked into cross-species infectivity. In horses, S. equisimilis is generally considered infrequent or opportunistic, but has recently been isolated from cases of strangles-like disease. Rapid and sensitive diagnostic techniques could enable epidemiological studies and effective investigation of outbreaks involving these bacteria. In this study, PCR protocols previously described in cattle and in humans to detect the species S. dysgalactiae and the subspecies equisimilis were evaluated to detect specific sequences in equine samples. For this purpose, 99 monolateral nasal swabs were collected from horses from stud farms with a history of S. equisimilis infection and were tested blindly by bacteriological isolation and by single and duplex PCR. DNA for PCR was extracted both from the colonies grown on agar media and from enrichment broth aliquots after incubation with nasal swab samples. S. equisimilis was identified by bacteriological isolation in 23 out of 99 swab samples, and PCR assays on these colonies were fully concordant with bacteriological identification (kappa statistic = 1.00). In addition, PCR of the enrichment broth aliquots confirmed the bacteriological results and detected S. equisimilis in 6 samples more than the bacteriological examination (kappa statistic = 0.84). The PCR protocols appeared to be reliable for the rapid identification of S. equisimilis in equine nasal swab samples, and could be useful for microbiological diagnosis.