Two KP1(–) strains of Trypanosoma rangeli (SC-58, SC-61) isolated from the wild rodent Phyllomys dasythrix from Santa Catarina (Brazil) were compared with some KP1(+) reference stocks from different Latin America countries, and also with Trypanosoma lewisi. The strains were analyzed by some morphological and biological features, and by biochemical and molecular techniques. The mean total length (TL) of the bloodstream trypomastigotes of T. rangeli varied between 31.3–33.0 μm, and those of T. lewisi (adult forms) was 28.2 μm, values within the variation known for each species. In T. rangeli KP1(+) and T. lewisi, the nucleus was located in the anterior portion of the body, with nuclear indexes (NI) ≥ 1.2, as typically described for both species. Differently, most trypomastigotes of the KP1(–) stocks presented NI ≤ 1.0. Another striking feature of the KP1(–) strains was their very fastidiously growth in axenic cultures when compared with the KP1(+) stocks and T. lewisi. Three isoenzyme loci (MDH, IDH and PGM) clearly distinguished T. rangeli and T. lewisi, and the distinction between the KP1(+) and KP1(–) strains was possible at MDH, PGM and GPI loci. All T. rangeli strains presented the typical 760 bp amplicon derived from their KP2 minicircles. However, the KP3 products of the KP1(+) strains were a single large band (~330bp), whereas those of the KP1(–) had two distinct bands (350 and 300 bp). T. lewisi presented 700 and 400 bp amplicons, as previously reported. The peculiarities of T. rangeli isolates from P. dasythrix corroborate a possible speciation process within this taxon.