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Callose is an important biopolymer of β-1,3-linked glucose units involved in different phases of plant development, reproduction and response to external stimuli. It is synthesized by glycosyltransferases (GTs) known as callose synthases (CalS) belonging to family 48 in the Carbohydrate-Active enZymes (CAZymes) database. These GTs are anchored to the plasma membrane via transmembrane domains. Several genes encoding CalS have been characterized in higher plants with 12 reported in the model organism <i>Arabidopsis thaliana</i>. Recently, the <i>de novo</i> transcriptome of a fibre-producing clone of stinging nettle (<i>Urtica dioica</i> L.) was published and here it is mined for <i>CalS</i> genes with the aim of identifying members differentially expressed in the core and cortical tissues of the stem. The goal is to understand whether specific <i>CalS</i> genes are associated with distinct developmental stages of the stem internodes (elongation, thickening). Nine genes, eight of which encoding full-length <i>CalS</i>, are identified in stinging nettle. The phylogenetic analysis with CalS proteins from other fibre crops, namely textile hemp and flax, reveals grouping into 6 clades. The expression profiles in nettle tissues (roots, leaves, stem internodes sampled at different heights) reveal differences that are most noteworthy in roots vs. leaves. Two <i>CalS</i> are differentially expressed in the internodes sampled at the top and middle of the stem. Implications of their role in nettle stem tissue development are discussed.