Populations of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have been declining for multiple decades because of a variety of stressors associated with anthropogenic habitat disturbance followed by riparian sedimentation. The influence of parasite-associated morbidity and mortality on wild hellbender populations is poorly understood. Research has detected widespread trypanosome infection in hellbenders in Virginia, US, with no other reported detections within the eastern hellbender's extensive range. In our study, trypanosomes mostly closely resembling Trypanosoma cryptobranchi were observed in a blood smear of a hellbender from Tennessee during a population survey. Banked whole blood from this hellbender along with 51 other hellbenders was molecularly tested for the 18s rRNA gene of amphibian trypanosomes using newly designed PCR primers. In total, 3/52 (5.8%) hellbenders were PCR- and sequence-positive for trypanosomes. This is the first report of the partial 18s rRNA sequence from hellbender trypanosomes from North America. Further research into trypanosome epidemiology and hellbender health implications is warranted.