AbstractHaematophagous insects can rely on specialized host‐seeking behaviors to locate hosts. Some frog‐biting flies, for example, eavesdrop on the conspicuous acoustic signals produced by male frogs and toads. Using such auditory cues to locate a host imposes an additional challenge: how to recognize appropriate sounds when different frog species produce calls with varying acoustic properties. The limited knowledge of antennal hearing in dipteran insects hinders our ability to understand how eavesdropping flies detect and recognize frog calls. Behavioral studies suggest that frog‐biting flies use broad acoustic templates to detect and recognize their victims. Here, we use within‐species call variation to examine the acoustic preferences in frog‐biting flies. Specifically, we examine the attraction of frog‐biting mosquitoes (Uranotaenia spp.) and midges (Corethrella nippon) to the calls of a Japanese treefrog, the Ryukyu Kajika frog (Buergeria japonica), on Iriomote Island, Japan. Male Ryukyu Kajika frogs produce two call types. While both calls have a high frequency peak (3 kHz), the first call type (Type I) also contains a lower frequency peak (1.8 kHz) absent in the second call type (Type II). Using field phonotaxis experiments we found that Type I calls are more attractive to both frog‐biting mosquitoes and midges. Thus, our results suggest that the frog‐biting Nematoceran flies in this community are biased towards the acoustic properties of Type I calls. We discuss this finding in the context of the evolution of antennal hearing in flies.