The placenta, a fetal endocrine organ, is composed of subpopulations of trophoblasts, including cytotrophoblasts, and syncytiotrophoblasts. Trophoblastic populations can be distinguished based upon their expression of cytokeratin. The purpose of the current study was to develop an immunohistochemistry (IHC) method to identify trophoblasts selectively in frozen feline placental tissue using antibodies specific for cytokeratin. The mouse monoclonal antibody anti-human pan-cytokeratin AE1/AE3 and a commercial detection system were used. Nonspecific immunoreactivity was encountered that could not be eliminated with altered blocking methods. The nonspecific reactivity was attributed to the goat anti-mouse/rabbit immunoglobulin G (IgG) peroxidase polymer included in the commercial kit. Alternatively, a polyclonal rabbit anti-cow cytokeratin wide spectrum screening antibody with goat anti-rabbit IgG polyclonal secondary antibody was used to detect cytokeratin in feline placental tissue. The IHC procedure eliminated nonspecific immunoreactivity while specifically labeling cytokeratin. This new approach provides an IHC method to identify trophoblasts specifically in feline placenta.