We analyzed variation in the advertisement calls among three localities of Eleutherodactylus glamyrus, a frog endemic to the Sierra Maestra mountains in Eastern Cuba. We assessed the levels of within-male variation of each call property and the influence of temperature and size of calling male on acoustical features. The typical single-note advertisement call of the species was described using temporal and spectral parameters. Rise time and frequency modulation were highly variable within individuals, whereas dominant frequency and call duration were the most stereotyped properties. Call rate showed an intermediate level of variation. Air temperature strongly influenced call rate and call group duration. Snout–vent length (SVL) strongly influenced dominant frequency and rise time. Localities differed in call rate, call duration, and rise time. This acoustic differentiation might be the result of past divergences arising from a distribution gap between the two mountain massifs surveyed. Because acoustical features are increasingly used in interspecific or intraspecific comparisons in the genus Eleutherodactylus, we encourage researchers to assess whether temperature or SVL have sufficiently important effects on their data and use statistical procedures to remove these confounding factors. The multinote call of the species is quantitatively described for the first time. This call type resembles that of other members of the genus and probably carries an aggressive message.