Objective: To explore the co-detection of natural infection of Trypanosomatidae parasites such as Leishmania and Crithidia in reservoir hosts of leishmaniasis. Methods: Rodent populations were monitored in two endemic foci of cutaneous leishmaniasis of Fars province, southern Iran from March to October 2016. Rodents were trapped alive in several parts of Shiraz and Kharameh cities. Afterwards, their organs were prepared for detection of Leishmania and Crithidia species by molecular, microscopic, and culture methods. Results: Totally, 115 rodents of five species; Tatera indica (T. indica) (85), Rattus rattus (12), Meriones libycus (9), Mus musculus (7), and Rattus norvegicus (2), were trapped alive and their tissue samples were examined using microscopic, cultivation, and molecular assays. Overall, 59 (51.3%) rodents were positive for Leishmania or Crithidia parasites. The highest rate (61.2%; 52/85) of Leishmania infection was related to the T. indica population. The cultivation, and molecular observations showed that two (2.4%; 2/85) of T. indica (foot-pad, and spleen samples) were positive to Crithidia. Conclusions: This is the first report of Crithidia infection in T. indica in Iran. Consequently, more epidemiological and ecological studies are needed to understand the role of Crithidia and Leishmania in T. indica.