Hybridization using kDNA and rDNA sequences as probes was performed to study phylogenetic relatedness of different species of trypanosomatids. Using this approach, we identified five organisms which had been classified as Phytomonas and Herpetomonas that were more closely correlated to each other phylogenetically than to any other species or isolates from either genera. These findings raise doubts about the validity of the current classification of Trypanosomatidae. Finally, we demonstrated the usefulness of kDNA sequences as an alternative to genomic sequences in obtaining phylogenetic information on trypanosomatids.