The aim of this work was to isolate starch from different parts of rye grain by a traditional and newly developed enzymatic method, and to compare the physical, chemical and molecular properties of isolated starches, especially focusing on susceptibility to resistant starch (RS) formation. Enzymatic treatment provided a high yield of starch which differed in granularity depending on the parts of grain used for isolation. A share of extreme size granules in starches have a strong impact on swelling and pasting properties. The differences could be explained by molecular properties of starch fractions in the examination of which HPSEC with post-column derivatization was especially helpful showing higher dispersity and molar mass of amylose in starches obtained by the enzymatic method. The derivatized SEC profiles of RS have not been reported earlier. Starches isolated by enzymatic method with a high share of small starch granules were less susceptible to a RS formation.