Parasites of the genus Trypanosoma are unicellular flagellated microorganisms of the Trypanosomatidae. This study describes an isolate of the genus Trypanosoma naturally infecting Rhipicephalus microplus ticks, characterized through molecular, morphological and biological analysis. Trypanosome cultures, designated strain P1RJ, were obtained by isolation from R. microplus haemolymph in cultures of the tick cell line IDE8. After isolation, strain P1RJ grew well axenically in L15B medium at temperatures of 30, 32 and 34 °C. The new trypanosome remained stable in axenic culture over 14 passages in L15B at 30 °C and was successfully cryopreserved and resuscitated. Morphometric analysis was performed on randomly selected developmental forms. 18S rRNA and 24Sα rDNA sequence analyses confirmed that strain P1RJ is a new species of the genus Trypanosoma. The nucleotide sequences described were submitted to Genbank. Pathogenicity, involvement in vertebrate hosts, epidemiology, developmental cycle and transmission mechanisms of strain P1RJ are still unknown. Therefore, more studies will be necessary to determine life cycle aspects of this trypanosome, for which we propose the name Trypanosoma rhipicephalis sp. nov.