PCR-based methods to amplify the 3' untranslated region (3'-UTR) of the heat shock protein 70 (type I) gene (HSP70-I) have previously been used for typing of Leishmania but not with Leishmania (Mundinia) martiniquensis and L. (Mundinia) orientalis, newly identified human pathogens. Here, the 3'-UTRs of HSP70-I of L. martiniquensis, L. orientalis, and 10 other species were sequenced and analyzed. PCR-Restriction Fragment Length Polymorphism (RFLP) analysis targeting the 3'-UTR of HSP70-I was developed. Also, the detection limit of HSP70-I-3'-UTR PCR methods was compared with two other commonly used targets: the 18S small subunit ribosomal RNA (SSU-rRNA) gene and the internal transcribed spacer 1 region of the rRNA (ITS1-rRNA) gene. Results showed that HSP70-I-3'-UTR PCR methods could be used to identify and differentiate between L. martiniquensis (480-2 bp) and L. orientalis (674 bp) and distinguished them from parasites of the subgenus Viannia and of the subgenus Leishmania. PCR-RFLP patterns of the 3'-UTR of HSP70-I fragments digested with BsuRI restriction enzyme successfully differentiated L. martiniquensis, L. orientalis, L. braziliensis, L. guyanensis = L. panamensis, L. mexicana = L. aethiopica = L. tropica, L. amazonensis, L. major, and L. donovani = L. infantum. For the detection limit, the HSP70-I-3'-UTR PCR method could detect the DNA of L. martiniquensis and L. orientalis at the same concentration, 1 pg/μL, at a similar level to the SSU-rRNA PCR. The PCR that amplified ITS1-rRNA was more sensitive (0.01 pg/μL) than that of the HSP70-I-3'-UTR PCR. However, the sizes of both SSU-rRNA and ITS1-rRNA PCR amplicons could not differentiate between L. martiniquensis and L. orientalis. This is the first report of using HSP70-I-3'-UTR PCR based methods to identify the parasites causing leishmaniasis in Thailand. Also, the BsuRI-PCR-RFLP method can be used for differentiating some species within other subgenera.