Anchovy is one of the economically important fish in the western part of the northern coast of Central Java. The production of anchovies extends along the coast from Brebes Regency to Batang Regency. Therefore, this study aims to determine the diversity of anchovies on the northern coast of Central Java (Brebes, Tegal, Pemalang, Batang, and Kendal), using a molecular approach. This study was conducted from April to September 2019 in the Laboratory of Tropical Marine Biotechnology, Faculty of Fisheries and Marine Science, Universitas Diponegoro. The descriptive exploratory method was applied in this study with a random sampling technique, while the cytochrome oxidase I (COI) gene was used for molecular identification. An anchovy sample molecularly identified from each region was found by sampling, including BB 3, PML 2, BTG 1, BTG 2, BTG 4, TGL 2, TGL 4, KDL 1, and KDL 4. The base-pair length of 10 anchovy samples from the COI gene amplification result was 600-700 bp. The sequencing and alignment results of the BLAST analysis showed that the ten anchovy samples were included in the type of