The mechanisms underlying bast fibre differentiation in hemp (Cannabis sativa L.) are largely unknown. We hybridised a cDNA microarray with RNA from fibre enriched tissues extracted at three different positions along the stem axis. Accordingly, we identified transcripts that were enriched in tissues in which phloem fibres were elongating or undergoing secondary wall thickening. These results were consistent with a dynamic pattern of cell wall deposition involving tissue specific expression of a large set of distinct glycosyltransferases and glycosylhydrolases apparently acting on polymers containing galactans, mannans, xylans, and glucans, as well as raffinose-series disaccharides. Putative arabinogalactan proteins and lipid transfer proteins were among the most highly enriched transcripts in various stem segments, with different complements of each expressed at each stage of development. We also detected stage-specific expression of brassinosteroid-related transcripts, various transporters, polyamine and phenylpropanoid related genes, and seven putative transcription factors. Finally, we observed enrichment of many transcripts with unknown biochemical function, some of which had been previously implicated in fibre development in poplar or cotton. Together these data complement and extend existing biochemical models of bast fibre development and secondary wall deposition and highlight uncharacterised, but conserved, components of these processes.