The study of fear and aversive learning in non-mammalian vertebrates provides valuable insights into the evolutionary origins of emotional memory. Amphibians, with their relatively simple nervous systems, represent an ideal model for investigating these processes. Here, we examined the long-term retention of an aversive memory in a classical conditioning paradigm in the toad Rhinella arenarum using heart rate as a physiological dependent variable. Subjects were implanted with electrodes and exposed to conditioning sessions where a neutral saline solution (CS) was paired with a hypertonic aversive solution (US). Control animals were exposed only to the neutral solution. Anticipatory tachycardia (conditioned response) to the CS was used as an indicator of emotional learning. After acquisition, toads were tested for retention after 1, 4, 8, 16, and 32 days. Significant differences were found between the paired and control groups at all tested times (ANOVA, p < 0.05), indicating a persistent and robust conditioned response. Additionally, we used AgNOR histochemical technique (neuronal activity indicator) to obtain preliminary data on the brain structures involved in aversive memory processing. These findings demonstrate that R. arenarum is capable of forming and retaining long-term emotional memories expressed through autonomic responses. This evidence supports the idea that fear learning mechanisms are highly evolutionarily conserved.