Trypanosomatid mitochondrial DNA (kDNA) possesses thousands of copies of small circular molecules called minicircles. Due to a high level of nucleotide polymorphism among copies, sequence alignment for species or strain characterization is not appropriate. In this work we report dinucleotide absolute frequency as a method to analyze minicircle sequences heterogeneity in trypanosomatids. Using Trypanosoma rangeli and Leishmania guyanensis minicircles as example of sequence length heterogeneity, we show that dinucleotide frequency of minicircles whose length variation is less than to 10% is relatively constant. Dinucleotide frequencies in Leishmania genus point out three clusters of predominant dinucleotide profiles: GG/TT/TG for Old World species; ii) TT/AA/TA for New World species and iii) TT/GG(AA) TA(AT) for Sauroleishmania. Trypanosoma species displayed broad range composition and the highest frequency values. Their dinucleotide profile appears to be species specific, except for African trypanosomes which exhibit similar composition. The low number of sequences from Crithidia, Herpetomonas, Phytomonas and Wallaceina did not allow a generalized analysis, however some species present highly similar compositional profile, e.g., Wallaceina species. Distinct signatures for Trypanosomatidae family members can be generated by using values of absolute frequencies, range and composition of most/least frequent dinucleotides from minicircles. Each species can be graphically represented by a diagram of frequencies along with a box plot of summary statistics.2030-01-01