Flea beetles (Phyllotreta cruciferae and P. striolata) are the most damaging insect pests of canola, one of Canada’s most economically valuable crops. Chemical insecticides are currently the primary method of flea beetle control, but concerns about their impact on non-target organisms highlight the need for safer alternatives. RNA interference (RNAi) has been recognized for its ability to selectively kill insect pests by silencing target genes through sequence-specific exogenous double-stranded RNA (dsRNA). However, sequence similarity between pest and beneficial insects continue to pose risks about potential off-target effects. In this study, two dsRNAs targeting the UBE2L3 and Snf7 gene in P. cruciferae were evaluated for off-target effects in the predatory ground beetle Pterostichus melanarius. A bioinformatic analysis was used to identify the similarity between the dsRNA sequences and their respective orthologs in P. melanarius. Beetles were then injected with the dsRNAs, and relative gene expression and mortality were measured. Following injection of the UBE2L3 dsRNA, which shares 83.4% similarity and a 29 bp matching segment with P. melanarius, target gene expression was reduced by 69.9%. Conversely, injection of the Snf7 dsRNA, which only shares 62.6% similarity, did not induce gene knockdown. Despite the knockdown of UBE2L3, no significant mortality was observed in any treatment group, including the positive control, suggesting that an insufficient dose of dsRNA was used. These findings demonstrate that sequence similarity influences the off-target effects of dsRNA, which can be predicted using a bioinformatic assessment. Continued sequencing of beneficial insects is required so that dsRNA can be designed to have minimal similarity with other species to reduce off-target effects.