BACKGROUND: Sex determination is an essential issue in certain fish species, like sturgeons. During the rearing management, distinguishing between male and female fish is an important process for producing meat or caviar.OBJECTIVES: The current study aimed to find the feasibility of electric organ discharge (EOD) frequency ‎generating to introduce a novel method of sex determination in three freshwater species, namely the ‎black ghost knife fish (Aptronotus albifrons), goldfish (Carassius auratus), and the Sterlet ‎sturgeon (Acipenser ruthenus), as non-electric fishes.METHODS: In this regard, following sex detection, EOD frequency ‎of fishes were recorded via an oscilloscope. In addition, knife fish was used as a reference sample on which a lot of work has been done to evaluate the accuracy of the method and the use of the oscilloscope. Furthermore, the EOD frequency of male and female ‎Sterlet sturgeons was recorded and reported for the first time in this study.RESULTS: The frequency ‎magnitude was significantly different in genders of Sterlet (P<0.05) with higher EOD frequency in males ‎than in females. There was no recorded frequency in goldfish ‎male and female. EOD frequencies, which were recorded in male and female of black ghost fish, ‎represented significant differences compared with the other studies (P<0.05).CONCLUSIONS: The results achieved ‎in this study revealed that sex differences are one of the effective parameters in terms of EOD ‎frequency inequality; this non-invasive method can be utilized for sex identification of valuable ‎species, like sturgeon. However, it is required to gather advanced equipment and conduct further ‎research in physiology and biology of reproduction for accurate recording of electrical frequencies ‎and detection of their origins.