The European map butterfly () is a classic example of seasonal polyphenism because the spring and summer imagoes display two distinct morphological phenotypes. The light regime and temperature during larval and prepupal development determine whether or not the pupae commit to diapause and overwintering and thus whether spring or summer imagoes emerge. We used suppression subtractive hybridization to experimentally screen for genes that are differentially expressed in prepupae committed either to accelerated metamorphosis and egg production or diapause and overwintering. The range and ontology of the differentially expressed genes in prepupae developing from larvae exposed either to long-day (LD) or short-day (SD) conditions revealed fundamental differences. The SD prepupae preferentially expressed genes related to cuticle formation and immunity, reflecting the formation of a robust pupal exoskeleton and the upregulation of antimicrobial peptides as preparations for overwintering. One protein preferentially expressed in SD prepupae has a counterpart in that functions as a diapause duration clock. The differentially expressed genes in LD prepupae included several members of the families. We also observed the strong induction of different -like genes under SD and LD conditions which suggest a role in the developmental choice between seasonal phenotypes. Our transcriptomic data will facilitate the more detailed analysis of molecular mechanisms underlying seasonal polyphenism.