Background: The purpose of this research was to compare the clinical and epidemiological characteristics of patients with chronic Chagas disease with and without positive blood cultures for Trypanosoma cruzi. Methods: This was a retrospective longitudinal study that included 139 patients with chronic Chagas disease who underwent blood culture for T. cruzi. Blood cultures were performed using Novy-MacNeal-Nicolle medium enriched with Schneider's medium. Multivariate Cox proportional hazards regression analysis adjusting for age and sex was performed to identify if positive blood culture for T. cruzi was associated with all-cause mortality. Results: The blood culture positivity rate was 30.9%. Most patients were born in the Northeast and Southeast regions of Brazil. Patients with positive blood cultures were older (52±13 vs 45±13 y; p=0.0009) and more frequently women (72.1% vs. 53.1%; p=0.03) than patients with negative blood cultures. The frequency of patients with cardiac or cardiodigestive forms was higher among patients with positive vs negative blood cultures (74.4% vs 54.1%; p=0.02). A total of 28 patients died during a mean follow-up time of 6.6±4.1 y. A positive blood culture was associated with all-cause mortality (hazard ratio 2.26 [95% confidence interval 1.02 to 5.01], p=0.045). Conclusions: We found a higher proportion of patients with Chagas heart disease among patients with T. cruzi-positive blood cultures. A positive blood culture was associated with an increased risk of all-cause mortality. Therefore T. cruzi persistence may influence Chagas disease pathogenesis and prognosis.