In this study, a Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) method for genetic typing of Trueperella pyogenes, an opportunistic bacterial pathogen, was designed. The method optimization was performed for 37 clinical T. pyogenes strains isolated from various infections in different animal species. Optimal conditions for reliable and reproducible DNA fingerprinting were determined according to the modified Taguchi method. The developed method was assessed regarding its typeability, reproducibility, and discriminatory power using the Hunter’s and Gatsons’ index of discrimination. A high degree of genetic diversity was shown between the studied strains, which represented 31 genotypes. The generated RAPD profiles were relatively complex and simultaneously easy to interpret due to the wide size range of amplicons. The discriminatory index of the designed method was sufficiently high; thus, only strains epidemiologically related displayed identical RAPD genotypes. In conclusion, the DNA fingerprinting of T. pyogenes by the developed RAPD-PCR method is a reliable typing tool that may allow a better understanding of the epidemiology as well as pathogenesis of infections caused by this pathogen.