A positive Trypanosoma cruzi polymerase chain reaction (PCR) is associated with a worse prognosis in patients with chronic Chagas disease (CD). To study the association of clinical, electrocardiographic, and echocardiographic characteristics and biomarker blood levels with positive T. cruzi PCR in chronic CD. This is a single-centre observational cross-sectional study. Positive T. cruzi PCR association with clinical, electrocardiographic, and echocardiographic characteristics, and biomarker blood levels were studied by logistic regression analysis. p values < 0.05 were considered significant. Among 333 patients with chronic CD (56.4% men; 62 ± 10 years), T. cruzi PCR was positive in 41.1%. Stepwise multivariate logistic regression showed an independent association between positive T. cruzi PCR and diabetes mellitus {odds ratio (OR) 0.53 95% confidence interval (CI) 0.30-0.93; p = 0.03}, right bundle branch block OR 1.78 (95% CI 1.09-2.89); p = 0.02, and history of trypanocidal treatment OR 0.13 (95% CI 0.04-0.38); p = 0.0002. Among patients with a history of trypanocidal treatment (n = 39), only four (10%) patients had a positive T. cruzi PCR. Among several studied parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with positive T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR.