It was known previously that overwintering larvae of the beetle Dendroides canadensis produce antifreeze proteins (DAFPs) consisting of a family of 12 similar proteins, and based on sequence variations the DAFPs may be separated into three groups. DAFPs were known to be present in hemolymph, midgut fluid and in/on epidermal cells located immediately under the cuticle. However, only DAFPs-1, 2, and 4 were known to be present in the hemolymph, leaving the location of the others unknown. In this study, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry of hemolymph confirmed the presence of DAFPs-1, 2, and 4 (Group I), plus a protein consistent with the mass of DAFP-6 (Group I). Also, a review of older data revealed the co-purification of DAFP-6 along with DAFP-4 in hemolymph. However, none of the other DAFPs (Groups II and III) were present in hemolymph. In contrast, mass spectrometry of midgut fluid demonstrated the absence of DAFPs-1, 2, 4, or 6, however, proteins consistent with the masses of all, or a subset of, Groups II and/or III were present. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of all 12 DAFPs were present in the fat body. However, consistent with the MALDI-TOF data, only Groups II (8, 9, 10, 11) and III (3, 5, 7, 12) transcripts were found in midgut epithelia. RT-PCR of epidermal tissue identified dafps- 4, 6, 8 and 11 (and sometimes 1 and/or 2) as the major transcripts. These data suggest that various DAFPs may have evolved to function best in certain sites.