Abstract: The aim of this study was designing a diagnostic kit for yersiniosis in the trout fish in Iran. Colonies of Yersinia ruckeri were collected from culture medium and a suspension was prepared in a lysing solution. DNA was extracted through a boiling and phenol chloroform method. Two primer sets targeting bacterial 16S rRNA and trout 18S rRNA. Polymerase chain reaction products were separated by gel electrophoresis. Among 20 suspected samples tested two samples were positive for both host and bacterial PCRs indicating the positive Y. ruckeri infection and remaining 18 samples were negative for pathogen. The performance of PCR reactions in negative samples were confirmed from amplification of internal control reactions targeting host. A PCR based diagnostic kit with an internal control was prepared for detection of Yersinia ruckeri in rainbow trout fish.