Characteristics of whistles may be used to study differentiation in dolphins to complement morphological and genetic studies. The whistles of four populations of Chinese humpback dolphins (Sousa chinensis chinensis (Osbeck, 1765)), one population of Taiwanese humpback dolphins (Sousa chinensis taiwanensis Wang, Yang, and Hung, 2015), and one population of Australian humpback dolphins (Sousa sahulensis Jefferson and Rosenbaum, 2014) were compared to determine if differences in whistles support current views of population structure and regional and species differentiation in the genus Sousa Gray, 1866. Acoustic features were extracted from whistles captured by broadband recording systems. Permutational MANOVAs were conducted to test for differences between populations, regions, and species. Random forest trees were also used to classify similar whistles. A significant amount of variation in acoustic features was explained by population (pseudo F [5,2742] = 191.66, p < 0.001), regional (pseudo F [3,2741] = 280.62, p < 0.001), and species (pseudo F [1,999] = 3.7, p < 0.05) differences in humpback dolphin whistles. Random forest trees correctly classified whistles into populations from 40% to 67%, regions from 51% to 80%, and species from 74% to 80%. Differences in whistles were consistent with the current ideas of population-, regional-, and species-level differences within the genus Sousa, based on morphological and genetic data, as well as geographic distance and barriers to movement.