Lactococcus garvieaeis the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout.L. garvieaehad been considered the only cause of lactococcosis for a long time; however,L. petauri, another species of the genusLactococcus, has lately been linked to the same disease. The genomes and biochemical profiles ofL. petauriandL. garvieaehave a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiateL. garvieaefromL. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. TheITSregion of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separateL. garvieaefromL. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely relatedL. garvieaeandL. petauriand it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.