Through digital sampling and resampling at 5,000 and 20,000 Hz of amplified mosquito flight sound, baseline separation was observed for flight tone frequency distributions of male and female Anopheles gambiae Giles, An. arabiensis Patton, An. merus Donitz, and An. melas Theobald. Males of the 4 species showed flight tones considerably higher than females. Up to 7 harmonics were measured for each species. Close correspondence for each individual mosquito of the means of the flight tone harmonics (corrected for harmonic number) demonstrated the accuracy and precision of the method. These data indicate that flight tone differences have been subjected to selection and may act as an isolating mechanism for mating or serve some other behavioral purpose in these mosquitoes. Individuals and swarms of sympatric species were distinguished from each other for both males and females, but the allopatric species, An. merus and An. melas, were indistinguishable.