The freeze‐intolerant insect, Choristoneura fumiferana (spruce budworm), produces multiple antifreeze protein (AFP) isoforms for protection during the overwintering stage. We now report the cloning of AFP genes from insects; Afp‐Lu1 encodes a ≈ 9‐kDa AFP isoform, and Afp‐Iu1 encodes a ≈ 12‐kDa AFP isoform. Both CfAFP genes have similar structures with a single 3‐ to 3.6‐kb intron interrupting the coding region. The second exon of an additional CfAFP gene, 2.7a, encoding a new ≈ 9‐kDa isoform, was found 3.7 kb upstream of Afp‐Lu1 and demonstrates that some AFP family members are linked in tandem. This gene appears to encode an AFP with 68–76% identity to previously isolated CfAFPs. With its eight Cys residues necessary for disulfide bonding and five perfectly conserved ‘Thr button’ (Thr‐Xaa‐Thr) ice‐binding motifs, it can be modeled as a functional AFP. Southern blot analysis shows that there are ≈ 17 genes in this AFP family, with each of the isoforms represented by two to five gene copies. Transcript accumulation from Afp‐Lu1 and Afp‐Iu1 (or closely related genes) was maximal during the overwintering stage, while 2.7a transcripts were only detected in first instars, larvae that are normally found only in the summer. Contrary to expectations, this differential expression demonstrates that CfAFP gene family transcripts are primarily regulated during development, rather than by seasonally low temperatures.