The persistence of Trypanosoma cruzi in seropositive individuals, previously diagnosed as chronic chagasic patients (CCP), was detected for the first time in biopsies taken from gingival inflammatory foci processed by polymerase chain reaction (PCR). Seven out of 31 (22.5%) gum samples from selected unquestionably CCP showing different degrees of gingival inflammation revealed T. cruzi-DNA using 3 specific PCR assays. All the included CCP had been diagnosed in previous studies carried out over the last 19 years. Samples of inflamed gums were recently taken from the indicated patients at: an outpatient hospital cardiac unit; a village where Chagas disease is endemic; and a specialized diagnostic research center, showing molecular evidence of parasite persistence in 17.6%, 42.8% and 14.3% of them, respectively. The relatively frequent parasite persistence, demonstrated here in oral inflammatory processes of treated and/or untreated patients bearing long term T. cruzi-infection, suggests the establishment of secondary small foci for the maintenance of hidden or inapparent chagasic infection. The easy and low-risk, non-invasive method to get the sample may add the use of gingival biopsy as a potential alternative diagnostic tool to confirm T. cruzi-infection in CCP. The significance of T. cruzi persistence as a primary cause of chronic Chagas disease and the proposal of this mechanism to explain the pathogenesis in CCP are considered.