Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis. Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V.) braziliensis in the state of Rondônia. L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River. Leishmaniasis is considered a neglected disease with 1.5 million new cases of cutaneous leishmaniasis (CL) occurring each year. In the Amazon region and in the Americas in general, ML is caused by Leishmania (Viannia) braziliensis, though in rare cases it has been related to other species. ML, which is associated with inadequate treatment of CL, normally manifests itself years after the occurrence of CL. Clinical features evolve slowly and most often affect the nasal cavity, in some cases causing perforation, or even destruction, of the septum. Diagnosis is made using the Montenegro skin test, serology and histopathology of the patients' mucosal tissues, or by isolation of the parasites. PCR is the best way to identify the species of leishmaniasis and is therefore the diagnostic method of choice. This paper describes 46 cases of ML and their geographical origin, 30 cases associated with L. (V.) braziliensis and 16 with L. (V.) guyanensis. The species of leishmaniasis was identified using mucosal biopsies taken from Amazonian patients who were diagnosed and treated for ML in the tertiary care unit, in Manaus, Amazonas state, Brazil. This is the highest number of ML cases caused by L. (V.) guyanensis that has ever been reported.