In this research letter, we report the development and validation of a new subset of fluorescence‐based CRISPR interference (CRISPRi) tools for our scientific community. The pJL series is directly derived from the original pIRL CRISPRi vectors and conserves all the elements to perform inducible targeted gene repression. These vectors carry two distinct fluorescent markers under the constitutive promoter psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the glycopeptidolipid translocase mmpL4b and the essential genes rpoB and mmpL3 . Finally, we describe an efficient single‐step procedure to co‐transform mycobacterial species with this integrative genetic tool alongside episomal vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research.