Abstract Diapause-associated gene expression was studied in Drosophila triauraria using subtractive hybridization. Two genes that were shown to be upregulated in diapausing flies by Northern hybridization have similarity to genes encoding antifungal peptides of Drosophila melanogaster, members of the drosomycin family (drosomycin, CG10812, CG10813, CG10815 and CG11520). In addition, a signal peptide and Knot 1 domain are shared with them. The genes cloned from D. triauraria are tentatively named drosomycin-like. However, the similarities between drosomycin-like in D. triauraria and the members of the drosomycin family in D. melanogaster are quite lower than those between other homologous genes in these species. In addition, neighbor-joining analysis revealed that drosomycin-like in D. triauraria is not closely related to known members of the family in D. melanogaster. Thus, it is most plausible that drosomycin-like is not a D. triauraria counterpart of known members of the family, but a novel member belonging to the family. The drosomycin-like gene is expected to have a few copies, because at least two sequences having unique 3′-ends were obtained in RACE, and multiple bands were observed in Southern hybridization. However, these sequences from RACE had the same ORF. Probes for genes encoding additional antimicrobial peptides were used to evaluate expression during diapause. Like drosomycin-like, drosomycin was upregulated during diapause, but defensin and drosocin were not.