To analyze the molecular basis of migratory behavior in the monarch butterfly (Danaus plexippus), we generated an expressed sequence tag (EST) resource for the butterfly brain. Migratory and “summer” (non-migratory) butterflies were collected from the wild, and whole brains were dissected. mRNA was extracted, and a normalized unidirectional library was constructed at the Keck Center. The library was subsequently subtracted and 21,596 cDNA clones were partially sequenced, with an average reading length of 741 nt with base quality of at least 20. The sequences were assembled into 9484 unique sequences that were tested for similarity with sequences in the Drosophila, mosquito (Anopheles gambiae), honey bee (Apis mellifera), and commercial silkworm (Bombyx mori) annotated genome databases and non-redundant from NCBI. There were 6274 non-overlapping hits with the other databases. Transcriptional profiling of non-migratory and migratory butterflies has been initiated.