Here, we have investigated how a subset of box C/D snoRNAs can modify multiple nucleotides within the rRNA target region using a single guide and C΄/D΄ motif. Our data indicate that this is primarily achieved using unusual C΄/D΄ motifs. The established rule is that the position in the rRNA five nucleotides upstream of the D or D΄ box is methylated (Figure 1A) (25). However, the U18 and snR48 C΄/D΄ motifs, when used in an artificial snoRNA construct, direct the modification of multiple sites in the target region, while the snR13 C΄/D΄ motif directed the modification of the position in the rRNA six nucleotides upstream of the ‘canonical’ D΄ box. The snR48 snoRNA contains a long and unusual D΄ box that lacks a sequence comparable to the consensus and we predict contains both a four and six nucleotide long D΄ motif. In snR13 and U18, the D' motif lacks the C at the 5΄ end (CUGA) which our data indicate is not essential for the level of modification but is important for the correct selection of the target nucleotide. We propose that the snR13 and U18 snoRNAs contain two, overlapping four and five nucleotide long D΄ boxes. The five nucleotide long D΄ boxes include the last (3΄) nucleotide of what is considered the guide region (Figures 2B and 5B). We showed that having a C at the last nucleotide of the guide, which is found naturally in snR13, effectively lengthens the D΄ box by one nucleotide and causes methylation of the target site at a position one nucleotide further away in the target rRNA. Therefore, a C at this position in the guide would be predicted to interfere with target specificity. This is supported by our observation that a C at this position is significantly under-represented in the active guide/D΄ motifs of yeast snoRNAs. The D΄ box has always been considered to function as part of a ‘molecular ruler’ in defining the nucleotide for 2΄-O-methylation. Our data, however, indicate that the 5΄ end of the D΄ box, in particular, is the element that determines which nucleotide will be modified. We propose that there are two, overlapping D΄ boxes in snR13, U18 and snR48, which form alternative interactions with the C’ box, and depending on which D΄ box is recognised/used, this determines which nucleotide is modified. One explanation for the ability of these snoRNAs to direct the modification of multiple sites in target region is that each snoRNA can form two structural isoforms, with each capable of modifying a single, distinct, nucleotide. While we believe that the structural isoforms will be stable, especially when the proteins are bound, and two sub-populations of snoRNP exist. However, we cannot rule out the possibility that the snoRNP can alternate between the two conformations.