When confronted with a predator, many mammalian species emit vocalizations known as alarm calls. Vocal structure variation results from the interactive effects of different selective pressures and constraints affecting their production, transmission, and detection. Body size is an important morphological constraint influencing the lowest frequencies that an organism can produce. The acoustic environment influences signal degradation; low frequencies should be favoured in dense forests compared to more open habitats (i.e. the ‘acoustic adaptation hypothesis'). Such hypotheses have been mainly examined in birds, whereas the proximate and ultimate factors affecting vocalizations in nonprimate mammals have received less attention. In the present study, we investigated the relationships between the frequency of alarm calls, body mass, and habitat in 65 species of rodents. Although we found the expected negative relationship between call frequency and body mass, we found no significant differences in acoustic characteristics between closed and open-habitat species. The results of the present study show that the acoustic frequencies of alarm calls can provide reliable information about the size of a sender in this taxonomic group, although they generally do not support the acoustic adaptation hypothesis. informacion[at]ebd.csic.es: García-Navas & Blumstein (2016). The effect of body size and habitat on the evolution of alarm vocalizations in rodents. Biol J Linn Soc DOI: 10.1111/bij.12789