Utilizing and conserving the genetic resources of grouper species depends on an understanding of fish genetic characterization. In this work, the genetic diversity and evolutionary relationships of several Serranidae species, which are significant both ecologically and economically, were evaluated using 16S rRNA gene. This study focused on the phylogeny relationships amongst two Serranidae species: Epinephelus aeneus (male), Epinephelus aeneus (female), and Mycteroperca rubra (male) collected from the Mediterranean Sea, Egypt, exactly from Port Said area. The average frequencies of adenine (A), thymine (T), cytosine (C), and guanine (G) were 29.7, 22.7, 24.1, and 23.7%, respectively. According to our results, the genetic relationships of two Serranidae species in the Mediterranean Sea are mostly related to each other in phylogeny analysis.