The echolocation calls of bats may contain a single acoustic element (the fundamental frequency or a harmonic) or multiple acoustic elements that may (or may not) include the fundamental element. We hypothesize that the detection of harmonics is affected by three factors: 1) species, 2) situation, and 3) recording quality. To test our hypotheses, we recorded and analyzed approximately 2,300 calls from 17 species and 1 subspecies in 6 families of bats using a 1-channel and 4-channel microphone array. The percentage of calls with multiple acoustic elements varied from 0 to 83% across species. Furthermore, recordings from a 4-channel microphone array (1 m tetrahedron arrangement) revealed that the percent of calls with multiple acoustic elements varied across channels by up to 50%, indicating the effect of bat position relative to the microphone. In some species, presence of multiple acoustic elements was predicted by call energy: calls with sufficient energy (threshold varied by species) had multiple acoustic elements above the noise floor of the recording system. In the remaining species that produced calls with multiple acoustic elements, we found two clusters of calls. In one cluster, the presence of multiple acoustic elements was predicted by received call energy. In the 2nd cluster, call energy was lower, and almost all calls included multiple acoustic elements. The detection of harmonics independent of recorded energy suggests the intriguing possibility that harmonics are used differently in these species. Finally, to test the effect of situation, we recorded the echolocation calls of big brown bats (Eptesicus fuscus) flying in three settings: an anechoic flight room, during roost emergence, and foraging in an open area. Call energy shifted to lower frequencies and fewer acoustic elements as the recording distance and the volume of the flight environment increased (i.e., as clutter decreased). Comparing flight room with foraging calls revealed that the second harmonic of open air foraging signals decreased by about 30 dB (relative to the fundamental). Overall, our results show that detection of echolocation signals with harmonics varied significantly across species. We also demonstrate that relative harmonic intensity varies according to the flight situation within a species, and when combined with the effects of call directionality and relative position of bat and microphone, these factors influence harmonic detection in echolocation recordings.