The main aim of the present investigation was to elucidate the efficacy of silver ion chromatography for selective separation of unsaturated (oleic, linoleic, and linolenic) fatty acids on a preparative scale. Accordingly, the present work was predominantly divided into two parts. In the first part, adsorption of oleic acid was carried out using commercially available ion exchange resins and silver-ion-loaded resins (R-Ag+) prepared in the laboratory from nonpolar and polar solvents in a batch mode. The maximum adsorption of oleic acid was found on R-Ag+ (454.55 g·kg−1) compared with other commercially available ion exchange resins from heptane at ambient temperature (303 K). The effect of temperature on the adsorption of oleic acid on R-Ag+ from heptane was investigated at 303, 313, and 323 K. The adsorption of oleic acid was favored at 303 K and decreased with a further increase in temperature. Experimental batch equilibrium data were modeled using the Langmuir and Freundlich isotherms. Further, thermodynamic parameters viz., ΔGads0, ΔHads0, and ΔSads0, were estimated. The negative values of ΔGads0 and ΔHads0 show that the adsorption of oleic acid on R-Ag+ was spontaneous and exothermic in nature. Based on the results obtained in the first part, the R-Ag+ resin was subjected to adsorption of fatty acids from industrial fatty acids mixture using heptane as a solvent at 303 K. A multicomponent Freundlich isotherm was used to model experimental batch equilibrium data. Linolenic acid and linoleic acid were preferentially adsorbed over oleic acid with selectivities of 1.40 and 1.16, respectively, from industrial fatty acids mixture.