Mycoplasma equigenitalium and Mycoplasma subdolum have been associated with infertility, endometritis, vulvitis and abortions in mares, and with reduced fertility and balanoposthitis in stallions. Despite their role in equine genital disorder, determinants of virulence and pathogenesis as well as factors provoking specific host immune responses have not been identified, so far. To establish the major immunogenic components of Mycoplasma (M.) equigenitalium and M. subdolum, antigen profiles of their type strains as well as 30 clinical isolates were compared by SDS-PAGE and immunoblot analysis using hyperimmune rabbit sera and equine sera from clinical cases. To define the major protein antigens of both mycoplasma species, detergent-phase fractionation with Triton X-114 was performed. Western blot analysis of 30 clinical isolates revealed a high similarity of their overall antigen profile with only slight differences. In contrast, monospecific polyclonal antibodies raised against detergent-phase proteins of the two mycoplasma species identified three prominent proteins (pST17, pST42, and pET45) undergoing variation in expression and size among clinical and clonal isolates.