The double features of lignin; its recalcitrance to cellulosic ethanol production and importance in xylem water transportation in terrestrial plants, has challenged scientists aiming to modify its content and composition because mostly such changes are related to alterations in plant development. The dirigent protein role in lignin polymerization remains elusive. In this study, two sugarcane varieties, early evaluated for a forage breeding program, were spatially analyzed for lignin (thioacidolysis, acid-soluble and insoluble Klason, ratio S/G by GC/MS). As a strategy to survey the lignin contrast among tissues, the rind and pith of the culm were analyzed separately from the top to the bottom and divided into three regions: mature, intermediary and immature. In addition, the dirigent proteins (DIRs) and monolignol biosynthesis enzyme transcriptional profile and co-expression were evaluated. From thirty-seven sugarcane DIRs found by ortholog analysis, 10 were evaluated by quantitative PCR (qPCR). ScDIR1 showed a positive correlation to S/G ratio, while ScDIR4, ScDIR5 and ScDIR7 showed a positive correlation to acid-soluble lignin content. The hierarchical clustering analysis revealed: ScDIR2 co-expressing with CcoAOMT5; ScDIR3 co-expressing with HCT6 and CcoAOMT9; ScDIR12 co-expressing with CcoAOMT6 and CAD1. These results suggest that DIRs are related to the lignin content and S/G ratio, which corroborate the DIRs action of stereoselective coupling of monolignols. ScDIRs transcripts are currently being evaluated by in situ hybridization for their co-localization with lignin accumulation in culm tissues. Furthermore, the functional analysis has been initiated.