Objective To assess heart rate variability (HRV) as a marker of autonomic nervous system disruption and its role in disease progression in dogs with Chagas disease (CD), and to evaluate arrhythmias and conduction abnormalities in symptomatic and asymptomatic groups. Methods A prospective observational study was conducted on dogs treated at a small animal hospital in central Texas from August to December 2023. Ambulatory 24-hour Holter monitoring was conducted to assess HRV metrics (proportion of pairs of successive NN intervals differing by ygt; 50 milliseconds and root mean square of the successive differences), arrhythmias, and conduction abnormalities. Heart rate variability parameters were categorized as high, normal, or low. Dogs were classified as symptomatic or asymptomatic on the basis of clinical presentation, and comparisons of HRV and ECG findings between groups were performed. Results 112 client-owned dogs with confirmed Trypanosoma cruzi infection were included. Of the 112 dogs, 46 (41.1percent-flag-change) were symptomatic and 66 (58.9percent-flag-change) were asymptomatic. Heart rate variability disruptions were observed in 63percent-flag-change of dogs, underscoring early and widespread autonomic dysregulation in T cruzi infection. Symptomatic dogs had more arrhythmias (1.54 vs 1.02) and a higher prevalence of second-degree atrioventricular blocks (0.19 vs 0.03), but HRV abnormalities were similar between groups. Conclusions Heart rate variability abnormalities were prominent across all dogs with CD, regardless of symptoms, suggesting their utility as early markers of autonomic and cardiac dysfunction. These findings highlight HRV’s potential for monitoring disease progression, particularly in asymptomatic dogs, supporting its inclusion in routine assessments for T cruzi infections.