Despite the successful use of numerous biomarkers of follicular development in mammalian species, markers for both adult and fetal horse ovarian development have yet to be investigated. In fetal development, primordial germ cells migrate from the yolk sac to the gonad during which mitotic division is initiated, resulting in an exponential increase in germ cells. Once established in the gonad, oogonia enter meiosis, arrest and a quiescent population of primordial follicles is generated. Activation of these follicles occurs during gestation and postnatally through local ovarian factors. The absence of gonadotropins and hormone receptors in both the somatic and germ cells of the horse ovary during fetal development means that these activated follicles are unable to survive. In this study, immunohistochemistry and immunoblotting were used to explore the validity of a number of mammalian ovarian developmental biomarkers. Zona pellucida glycoprotein 3 (ZP3), proliferating cell nuclear antigen (PCNA), cardiotrophin-like cytokine factor 1 (CLCF1) and forkhead box protein L2 (FOXL2) were assessed in an attempt to identify stages of follicular development in the fetal ovary. Both ZP3 and PCNA specifically bound to the oocyte. Cardiotrophin-like cytokine factor 1 (CLCF1) and forkhead box protein L2 (FOXL2) bound specifically to granulosa cells surrounding the oocyte, and strongly bound to pre-granulosa cells surrounding primordial oocytes. The verification of these proteins in the early equine ovary provides a basis for the visualisation of the primordial follicle pool in the horse and allows the reproductive lifespan of the animal to be estimated. This is particularly useful for further studies involving primordial follicle activation and regulation, which are important stages of folliculogenesis largely unexplored in the equid.