Spruce budworm (Choristoneura) species survive sub-zero winter temperatures by producing antifreeze proteins (AFPs) encoded by a multigene family of short and long isoforms. We report in this study the first analysis of antifreeze proteins from related Choristoneura sister species. The additional thirty amino acid insert found in the longer AFP isoforms maintains the proteins beta-helix and original fifteen amino acid (Thr-X-Thr) repeat motif. Analysis of the beta-helix region shows more divergent residues surround the conserved Thr residues. Maintaining the beta-helix structure and conserved Thr residues appear to be paramount for AFP function and surviving sub-zero winter temperatures. Two other species within the same lepidopteran clade, Ditrysia, do not appear to contain any AFP-like sequences.