The CRISPR-Cas system of bacteria and archaea provides resistance against mobile genetic elements. Whereas Type I and II CRISPR-Cas systems use CRISPR-derived crRNA guides to target complementary DNA, Type III-B systems have been demonstrated to perform RNA-guided RNA interference. Despite major differences in both target identity and cleavage mechanism, recent structural analyses have revealed that Type III complexes closely resemble the architecture of the Type I complexes (Cascade). Here we describe the elucidation of some key features of the interference mechanism of Type III-B Cmr complex of Thermus thermophilus (TtCmr). As with Cascade, it is demonstrated that mismatches between crRNA guide and a RNA target result in disrupted binding and cleavage by Cmr. Surprisingly, however, it was found that the seed region of the guide where mismatches are poorly tolerated resides at the 3’ end of the guide, and not at its 5’ end.