Bats (Chiroptera) comprise 30% of mammal species in Kruger National Park (KNP), yet their distribution and contribution to the environment are poorly understood. A preliminary survey of insectivorous bats (Microchiroptera) in KNP was conducted using ultrasonic detection. A total of 2,199 bat calls were recorded using a Pettersson D240x Ultrasonic Detector and were stored and displayed with SonoBat software. The software allowed for the visualization of call sonograms and the calculation of call parameters. Calls ranged in frequency from 10 kHz to 170 kHz. Bioacoustic identification of bats depends upon a library of known echolocation calls. No call library had been established for KNP, so bats were netted, identified based on morphology, and calls were recorded upon release. Hand release calls were obtained for eight of the nine species captured. The four parameters found to be most useful in distinguishing between the echolocation calls from the species captured were frequency at the knee, frequency at maximum amplitude, lowest frequency, and call duration. Bioacoustic surveys were conducted near Shingwedzi in the north of the park and Skukuza in the south. The calls of Nycteris thebaica, Pipistrellus rusticus, Scotophilus dinganii, and Scotophilus viridis were recorded near Shingwedzi. P. rusticus, S. dinganii, and S. viridis were identified by call in Skukuza. Neither geographic region (north or south) nor the presence of water had significant effect on the number of calls per hour recorded during acoustic sampling. Additional hand release calls are necessary to determine the distribution of Microchiroptera, especially that of endangered species, in KNP.