Geographic variation in acoustic characteristics of anuran advertisement calls could be an important indicator of species divergence due to its importance for species recognition. In this study, we evaluated the hypotheses of morphological by-product, local adaptation, and genetic drift to explain the geographic variation of the calls of the frog Eleutherodactylus nitidus. We compared six acoustic variables using ANOVA and linear discriminant analyses, as well as inter- and intra-population and intra-individual variation estimates to understand E. nitidus call variation. Recorded calls were corrected for temperature and body size to rule out the influence of these factors on call variation. The advertisement call of E. nitidus is a short whistle with an oscillating amplitude modulation that forms pulses. In total, we analysed 1676 advertisement calls from 66 individuals. We find that call duration, call amplitude, dominant frequency, pulses per call and highest pulse differ between populations, and such differences were independent of body size and temperature variation, ruling out the morphological by-product and local adaptation hypotheses. Additionally, the linear discriminant analysis indicates that geographic call variation depends principally on call amplitude. We suggest that the geographic distance between populations of E. nitidus yields genetic differences that account for differences in advertisement calls. [ABSTRACT FROM AUTHOR]