AbstractAcoustic communication of American Crocodiles (Crocodylus acutus) is relatively understudied. Our overall aim was to determine the acoustic structure of wild American Crocodile distress calls, distinguish call differences among size classes (hatchling, juvenile, sub‐adult, and adult), and investigate call production on a gradient of human disturbance. American Crocodile distress calls have strong frequency modulation and are comprised of multiple harmonics in a downsweeping pattern. Measured parameters (total duration, first quartile duration, maximal frequency, first quartile frequency, end frequency, slope of first quartile, slope of last quartiles) differed significantly among size classes (p < .05). Hatchling distress calls are higher in frequency and strongly modulated, whereas calls produced by sub‐adults and adults showed little modulation, are lower in frequency, and have greater overall duration. Proportion of crocodiles that produced distress calls during capture differed by size class and sampling location, particularly adult distress calls which are reported here to be produced with undocumented frequency. We determined that American Crocodiles of all size classes produce distress calls at varying rates among study sites. Our results demonstrate that American crocodiles produce distress call more frequently at sites with higher anthropogenic activity. Measured call parameters of juveniles and hatchling American crocodiles also varied among sites in relation to human disturbance. Calls recorded at sites of high anthropogenic impact have increased duration and less modulation which may adversely affect response to emitted distress calls. Proportional and call parameter variances suggest anthropogenic activity as a driver for increased call production and alteration of call parameters at high human‐impacted sites.