Abstract: Background and Objective: The DNA barcode can be used as a basis for determining the objectives and direction of development of conservation activities and can clarify differences and kinship relationships between species in the population. This study aimed to determine nucleotides sequences as DNA barcode for investigation of genetic diversity and phylogenetic from cuscuses in Maluku and Papua as well as other biogeography on COX3 gene with sequencing method. Materials and Methods: The genome DNA from 25 samples, extracted according to the Qiamp DNA protocol is the Mini Blood Kit (Qiagen) and is used as a template for COX3 gene amplification using PCR method. Results: Nucleotides alignment of Phalanger members found 23 nucleotides distinguishing and Spilocuscus members found 8 nucleotides distinguishing, which can be used as barcode. The phylogram tree was using Neighbor joining classified cuscus from Maluku and Papua in clade A (Phalanger) and clade B (Spilocuscus), respectively. Clade A and clade B subdivided into clade A1 (Phalanger from Maluku), A2 (Phalanger from Papua) and clade B1 (Spilocuscus from Maluku), B2 (Spilocuscus from Papua). Clade B2 subdivided into one subclade B2' be under cuscus from Halmahera dan Sentani, Jayapura Islands. Conclusion: It was concluded that identification of Spilocuscus and Phalanger members from Maluku, Papua, Halmahera and Sentani could be distinguished by COX3 gene. Spilocuscus maculatus from Halmahera is derived from Papua, it introduced in the colonization of the Austronesian tribes on the Island. S. maculatus from Halmahera and Sentani Islands have one nucleotide distinguish, that is sites position 354 which distinguishes the cuscuses with cuscus from Papua and Maluku.