It remains an enigma how the nervous system of different animal species produces different behaviors. We studied the neural circuitry for mating behavior inDrosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic modelD. melanogaster, in which the core courtship circuitry has been identified. We disrupted theD. subobscura fruitless(fru) gene, a master regulator for the courtship circuitry formation inD. melanogaster, resulting in complete loss of mating behavior. We also generatedfrusoChrimV, which expresses the optogenetic activator Chrimson fused with a fluorescent marker under the nativefrupromoter. Thefru-labeled circuitry inD. subobscuravisualized byfrusoChrimVrevealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification.SIGNIFICANCE STATEMENTHow did behavioral specificity arise during evolution? Here we attempted to address this question by comparing the parallel genetically definable neural circuits controlling the courtship behavior ofDrosophila melanogaster, a genetic model, and its relative,D. subobscura, which exhibits a courtship behavioral pattern unique to it, including nuptial gift transfer. We found that thesubobscura fruitlesscircuit, which is required for male courtship behavior, was slightly but clearly different from itsmelanogastercounterpart, and that optogenetic activation of this circuit inducedsubobscura-specific behavior, i.e., regurgitating crop contents, a key element of transfer of nuptial gift. Our study will pave the way for determining how and which distinctive cellular elements within thefruitlesscircuit determine the species-specific differences in courtship behavior.