Segara Anakan areas can be divided into three different regions according to their salinity. Salinity differences suggested that Commerson’s anchovy population in that area can be divided into three subpopulations due to genetic differences. Genetic differences among subpopulation can be assessed through a population genetic study using random amplified polymorphic DNA. This study aims to evaluate the genetic variation and differences of Commerson's anchovy (Stolephorus commersonnii) collected at three different water salinities in Segara Anakan estuary Cilacap Indonesia. Total genomic DNA was isolated using the Chelex method. Genetic diversity and differences were assessed using RAPD markers and were analyzed statistically using an analysis of molecular variance, as implemented in Arlequin software.  The results showed that high genetic diversity was observed within the subpopulations. However, no significant genetic differences were observed among subpopulations which indicate genetic similarity. A high number of offspring are likely to cause high genetic variation within subpopulations.  Adult and larvae migration is the cause of genetics similarity across Segara Anakan. Another impressive result is that water salinity did not affect the genetic characteristic of Commerson,s anchovy. Genetic similarity of Commerson’s anchovy indicates that Segara Anakan forms a single genetic conservation unit.