Behavioral barriers to gene flow often evolve faster than intrinsic incompatibilities, and can eliminate the opportunity for hybridization between interfertile species. While acoustic signal divergence is a common driver of premating isolation in birds and insects, its contribution to speciation in mammals is less studied. Here we characterize the incidence of, and potential barriers to, hybridization among three closely related species of grasshopper mice (genus Onychomys). All three species use long-distance acoustic signals to attract and localize mates; O. arenicola and O. torridus are acoustically similar and morphologically cryptic whereas O. leucogaster is larger and acoustically distinct. We used genotyping-by-sequencing (GBS) to test for evidence of introgression in 227 mice from allopatric and sympatric localities in the western United States and northern Mexico. We conducted laboratory mating trials for all species pairs to assess reproductive compatibility, and recorded vocalizations from O. arenicola and O. torridus in sympatry and allopatry to test for evidence of acoustic character displacement. Hybridization was rare in nature and, contrary to prior evidence for O. torridus/O. arenicola hybrids, only involved O. leucogaster and O. arenicola. In contrast, lab crosses between O. torridus

Onychomys genotypesZip file containing two data files with SNP genotypes from GBS and an Excel file with associated metadata and informationOnychomys_genotypes.zip