Regarding the increasing prevalence of antimicrobial resistance as a global menace, typing procedures are of great importance in epidemiological surveys. In the current study, interspecies differences of Yersinia ruckeri (Y. ruckeri) isolates were detected by antimicrobial susceptibility profiling and molecular traits to determine the relationship between isolates. The current study was conducted on 27 Y. ruckeri isolates collected from 40 infected rainbow trout (Oncorhynchus mykiss) with clinical enteric red mouth disease in Mashhad, Iran. Interspecies differences of all isolates were detected by antimicrobial susceptibility profiling using disk diffusion method, Repetitive Extragenic Palindromic PCR (REP-PCR), Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), and Random Amplification of Polymorphic DNA PCR (RAPD-PCR) to determine the relationship between isolates. Simpson’s diversity index was calculated for each typing technique. The most phenotype resistant was against ampicillin, chloramphenicol, and lincomycin. Furthermore, enrofloxacin and ciprofloxacin showed the highest activity (100%) against isolates. According to the results, the highest diversity index was observed in the REP-PCR method (D = 0.91). In conclusion, REP-PCR can be a powerful technique for epidemiological studies.