A systematic structural separation study was carried out for the separation of Pr(III) and Fe(Ill) from nitric acid medium with unsymmetrical diglycolamides (DGAs). Four kinds of diglycolamides, N,N'-di-methyl-N,N'-di-dodecyl diglycolamide (DMDDdDGA), N,N'-di-ethyl-N,N'-di-dodecyl diglycolamide (DEDDdDGA), N,N'-di-propyl-N,N'-di-dodecyl diglycolamide (DPDDdDGA) and N,N'-di-butyl-N,N'-di-dodecyl diglycolamide (DBDDdDGA) with different alkyl substituent were synthesized and characterized. The effect of the structure of extractant on the separation of iron and praseodymium was studied. A high separation coefficient of Pr(III) and Fe(III) was obtained by DMDDdDGA in a wide range of nitric acid concentration, which shows good application prospect in the recovery and purification of rare earth.