Postnatal development of vocalization has been studied in a small number of bats, not including the Geoffroy's bat, Myotis emarginatus. In the present study, we investigated vocalization development at a maternity roost of M. emarginatus in Kerend Cave in western Iran by sequential measurement of acoustic parameters in known-age neonates using mark-recapture sampling. Newborn pups of M. emarginatus produced both short and isolation calls. Duration of short calls of 1-day-old pups was on average 7.56 ± 0.05 ms and decreased during postnatal development to reach an adult-like duration of 2.78 ± 0.13 ms in the third week. Myotis emarginatus emitted various types of isolation calls, including classic, UP-tail-FM and CF-tail during postnatal growth. As bat pups grew, both short and isolation calls changed in their spectro-temporal structure. Discriminant function analyses showed that each bat pup has a vocal signature that facilitates mother-infant communication. The current study reveals that M. emarginatus pups are highly vocal at birth, but gradually elaborate their sounds with an increase in peak, start and end frequencies as well as with a decrease in call duration throughout the postnatal growth period.