<p>Gonadotropins of both pituitary and placental origin are complex glycoproteins essential for the regulation of reproductive physiology across mammalian species. While human chorionic gonadotropin (hCG) has been extensively characterized, pregnant mare serum gonadotropin (PMSG), also known as equine chorionic gonadotropin (eCG), remains the least understood among the major gonadotropic hormones. PMSG is unique because it exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) like biological activities in heterologous animal species, although it possesses only LH-like activity in equids. The molecular basis for this dual activity and the extent of its molecular heterogeneity has not been satisfactorily explained in existing literature. This study aimed to investigate the biochemical, physiological, and immunological characteristics of PMSG by developing a streamlined purification protocol and assessing the biological activity of separated fractions. PMSG was purified from pregnant mare serum (PMS) through a combination of ammonium sulphate fractionation, size exclusion chromatography on Sephadex G-100, and dye-ligand affinity chromatography using Cibacron Blue affinity column chromatography. The preparations were systematically analyzed using spectrophotometry, PMSG-specific ELISA, Sodium Dodecyl Sulphate –Poly Acrylamide Gel Electrophoresis (PAGE), Western blotting, and in vivo bioassays in Holtzman rats and Swiss mice. Our results demonstrate the successful isolation of a homogeneous PMSG preparation and the discovery of two distinct biologically active isoforms. One isoform exhibited high intrinsic dual FSH and LH activities, while a second, isoform behaved distinctively, exhibiting potent FSH-like activity with negligible LH-like activity. These findings provide significant insights into the structural–functional correlations of equine gonadotropins and the role of post-translational modifications in modulating hormone specificity.</p>