This investigation describes the comparative structural characterization of naturally-and synthetically-spun fibers of Bombyx mori fibroin. Wet spinning from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solutions was used to spin fibroin fibers with varying degrees of postspinning draw. Quantitative Raman spectroscopy showed that the secondary structure of fibroin changed dramatically from predominantly alpha-helical in HFIP solution to principally beta-sheet in the underdrawn synthetically-spun fiber. X-ray fiber diffraction showed that the underdrawn fibers were highly crystalline (>50%) with little or no preferential crystalline alignment. The addition of a postspinning draw caused the polypeptide backbone and beta-sheet crystals, which were formed largely by a methanol coagulation bath, to align with the fiber axis. The molecular and crystalline structures most similar to those of naturally-spun fibers were reproduced in synthetically-spun fibers with a minimum draw ratio of 2.5.