Background: Chagas disease is increasingly recognized in the southern U.S., where triatomine vectors transmit Trypanosoma cruzi among wildlife and domestic dogs with occasional vector spillover to humans. As in humans, clinical outcome in dogs is variable, ranging from acute death to asymptomatic infections or chronic heart disease. In order to characterize cardiac manifestations of T. cruzi infections, we tracked a cohort of naturally-infected dogs and a matched cohort of uninfected dogs. We hypothesized that selected measures of cardiac disease (abnormal rate, abnormal rhythm, and elevated cardiac troponin I (cTnI; a biomarker of cardiac injury)) would occur more commonly in infected than uninfected dogs matched by age, breed, sex and location. In addition to the clearly positive and negative dogs, we specifically tracked dogs with discordant test results across three independent serological assays to gather clinical data that might elucidate the infection status of these animals and inform the utility of the different testing approaches. Results: We placed an ambulatory ECG monitor (Holter) on 48 government working dogs and analyzed 39 successful recordings that met length and quality criteria from 17 T. cruzi-infected, 18 uninfected dogs and 4 dogs with discordant results. Overall, 76.5% of positive, 100.0% of discordant, and 11.1% of negative dogs showed > 1 ECG abnormality (p < 0.0001), and positive and discordant dogs had a higher mean number of different types of ECG abnormalities than negative dogs (p < 0.001-0.014). The most common cardiac abnormalities included supraventricular and ventricular arrhythmias and atrioventricular block. Positive dogs had higher serum concentrations of cTnI than both negative dogs (p = 0.044) and discordant dogs (p = 0.06). Based on dog handler reports, nearly all (4/5; 80%) dogs with reported performance decline or fatigue were T. cruzi-infected dogs.Conclusions: Further understanding cardiac manifestations in dogs naturally infected with T. cruzi is critical for prognostication, establishing a baseline for drug and vaccine studies, and better understanding of zoonotic risk.