The external anal sphincter (EAS) is important for the maintenance of bowel continence and may be compromised by a variety of neuropathic conditions. However, large animal models for the study of EAS functions have been sparse. The EAS guarding reflex was examined by electromyography (EMG) in neurologically intact rhesus macaques ( n = 6) and at 4-6 wk after a unilateral EAS denervation from an L6-S3 ventral root avulsion (VRA) injury ( n = 6). Baseline EAS EMG recordings were quiescent in all subjects, and evoked responses showed an initial large-amplitude EMG activity, which gradually returned to baseline within 1-2 min. At 4-6 wk postoperatively, the EAS guarding reflex showed a significantly reduced EMG response duration of 47 ± 15 s and area under the curve (AUC) of 0.198 ± 0.097 mV·s compared with the corresponding evoked EAS EMG duration of 102 ± 19 s and AUC of 0.803 ± 0.225 mV·s ( P < 0.05) in the control group. Detailed time- and frequency-domain analysis of the evoked EAS EMG responses for the first 40 s showed no difference between groups for the maximum amplitude but a significant decrease for the mean amplitude across the study period and an early AUC reduction for the first 10 s in the VRA injury group. Time-frequency analysis and power spectrum plots indicated decreased intensity and a narrower midrange of frequencies in the VRA injury group. We conclude that the EAS guarding reflex in rhesus macaques shows characteristic EMG features in control subjects and signs of partial target denervation after a unilateral L6-S3 VRA injury. NEW & NOTEWORTHY The external anal sphincter guarding reflex showed initial large-amplitude peaks and a gradual return to a quiescent baseline after a rectal probe stimulus in rhesus macaques. At 4-6 wk after a unilateral ventral root avulsion (VRA) injury, the electromyography duration, mean amplitude, and area under the curve measurements were decreased. Time-frequency analysis and power spectrum plots indicated decreased intensity and a narrowed midrange of frequencies in the VRA injury cohort.