Adult rat ultrasonic vocalizations (USVs) are a valuable tool for noninvasively assessing an animal’s emotional state. USVs are produced in 1 of 2 frequency ranges labeled as 22 kHz or 50 kHz vocalizations. One USV subtype within the 50 kHz call category, constant frequency 50 kHz (CF 50 kHz) calls, is not viewed as signaling an emotional state. The current study tested the hypothesis that CF 50 kHz calls are related to a mild negative affective state. In Experiment 1, diazepam (1, 2.5, or 5 mg/kg), or control injections were administered prior to receiving a sequence of mild footshocks (0.5 mA, 0.5 s). Subjects transitioned from producing CF 50 to 22 kHz USVs as footshocks were repeated; a pattern paralleled by a shift from rearing to increased time freezing. USV production was largely absent in the higher diazepam dose groups, whereas the 1 mg/kg dose attenuated CF 50 kHz USV production prior to and immediately following initial footshocks. The higher doses of diazepam similarly reduced rearing activity and overall freezing behavior. In Experiment 2, pre-exposure to the testing environment with or without access to palatable food elicited CF 50 kHz calls and rearing. During re-exposure to the test chamber the following day, CF 50 kHz USV production was reduced prior to footshock onset compared to the prior test day. The pattern of results support an association between CF 50 kHz USVs and a mild negative affective state; dissociating this call type may increase the sensitivity of behavioral measurements of emotion.