Echolocating bats use and adapt ultrasound pulses that vary in several parameters, one of them being the pulse’s source level, which is a measure of the emitted sound amplitude. This is a vital ecological parameter as it directly impacts the maximum distance over which bats can perceive targets in their environment, most importantly their prey. Different habitats present different sensing challenges for echolocation systems, and the quality and content of information derived from echolocation pulses reflect these environmental challenges. As such, echolocation pulses within or between species may vary from one habitat to the next due to variable selection pressure, resulting in local adaptation. Habitat is, therefore, a key component in shaping the evolution of echolocation. The Acoustic Adaptation Hypothesis (AAH) proposes that acoustic properties of the environment influence sound propagation and therefore the evolution of echolocation pulses. Here, we tested the AAH using multiple microphone arrays to measure the source levels of echolocation pulses of 14 bat species in bat assemblages across sites in six biomes in South Africa. Contrary to the AAH, our results revealed that bats in the same assemblage used different echolocation pulse source levels, frequencies, and duration resulting in different detection distances, which differ among bat assemblages occupying different sites. Furthermore, detection distance was species-specific and remained similar within species between assemblages; suggesting that species is a better predictor of detection distances compared to habitat as indicated by Miniopterus natalensis across all seven sites. KEYWORDS: adaptation, bat assemblages, detection distances, microphone arrays, selection pressure, source levels