ABSTRACT. This study characterizes the calling song and ultramorphology of the stridulatory file of two geographically isolated populations of the tropical bush cricket Eneoptera surinamensis (De Geer, 1773) from city of Foz do Iguaçu, state of Paraná, and town of Rio Claro, state of São Paulo, Brazil, distant 1,000 Km from each other. The teeth are shell-shaped, the larger ones are distributed in the medium region of the file, decreasing gradually in size towards the edges. Specimens from Foz do Iguaçu have a file with 82 ± 9.8 teeth, length=1.89 mm ± 0.15 with 43.76 ± 5.94 teeth per mm (n=15). Specimens from Rio Claro present a file with 87 ± 9.81, length=1.96 ± 0.19 mm with 44.52 ± 4.61 teeth per mm (n=15). Statistical differences found between the two populations are not significant. The calling song is an uninterrupted trill that alternates two sets of notes distinct for its temporal features.