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The two genetic software, DnaSP and NETWORK, is commonly used to analyse gene polymorphism and haplotype network which is useful for the understanding pattern of genetic variation and genetic relationship. In this study, the two software were applied to detect polymorphism of 16S sequence gene of Ophiocara paracephala from Tekolok Estuary (NTB, Indonesia) which has not been investigated. This study used a PCR method with 16Sar and 16Sbr primers. The results revealed two haplotypes and two variable sites with one parsimony informative within O.paracephala from Tekolok Estuary population. Next, if the 16S sequence data of O.paracephala from Tekolok Estuary was combined with the 16S gene data of O.paracephala from GenBank, three haplotypes were detected with five variables sites and one parsimony informative. This finding showed intra-population and intraspecific genetic variation of O.paracephala. In addition, haplotype network using NETWORK and a phylogenetic tree using Neighbor Joining and Maximum Likelihood methods exhibited that O.paracephala from Tekolok Estuary has a close genetic relationship with O.paracephala from Japan.