Morphometric characteristics of the periotic–timpanic bone complex in the middle ear of cetaceans, are effective characteristics in evaluating systematics. However, they have not been used for studies of geographic variation regarding dolphins of the genus Sotalia. This study aimed to compare the periotic–timpanic of Sotalia guianensis from four distinct locations, considered here as different operational taxonomic units, Amapá/Pará (AM/PA), Maranhão/Piauí (MA/PI), Ceará (CE), and Rio de Janeiro (RJ), using 21 morphometric measurements. Multivariate analysis showed significant distinction mainly between the units of northern (AM/PA and MA/PI) and south-eastern (RJ) Brazilian coast. The timpanic bone showed variation, reaching larger sizes in the Brazilian south coast unit, corroborating current molecular data on the geographic variation of S. guianensis.