Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR-based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S-23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false-positive results with non-L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.