Abstract: Cuscuses is marsupials animal (Phalangeridae), which has limited spread in Eastern Indonesia (Sulawesi, Maluku, Papua and Timor islands), Australia and Papua New Guinea. This study, the phylogenetic relationship of cuscuses from Papua and Maluku based on mitochondrial sequences of NADH dehydrogenase subunit 1 gene was investigated. Whole genome DNA was extracted from 22 tissue biopsy samples from Maluku (13 individuals) and Papua (9 individuals) according to the protocol of Qiamp DNA Blood Mini Kit (Qiagen) and then it was used as template for amplification of ND1 gene by using PCR method. The PCR product gives result nucleotides of 1152 bp and sequencing product gives result nucleotides of 956 bp of the ND1 gene for phylogenetic analysis. The genetic distance between Phalanger and Spilocuscus, 14.2% was found higher than the genetic distance between cuscus from Papua and Maluku 7.8%. The genetic distance within Phalanger was 3.7% and genetic distance within Spilocuscus was 1.3%. The genetic distance within Phalanger from Papua and Maluku was 1.3% and the average genetic distance within Spilocuscus from Papua and Maluku was 1.3%. The phylogram tree using Neighbor Joining classified cuscus from Papua and Maluku in clade A (Phalanger) and clade B (Spilocuscus), respectively. Clade A and B were further subdivided into clade A1 (Phalanger from Papua), A2 (Phalanger from Maluku) and clade B1 (Spilocuscus from Papua), B2 (Spilocuscus from Maluku). Spilocuscus genera members from Ternate and Sentani islands was found five nucleotides distinguishing compared to Spilocuscus from Papua and Maluku. It was concluded that identification of Spilocuscus and Phalanger members from Maluku, Papua, Ternate and Sentani could be distinguished by mitochondrial DNA sequencing of the ND1 gene.