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Transposable elements (TEs) are considered to be important components of the maintenance and diversification of genomes. The recent increase in genome sequence data has created an opportunity to evaluate the impact of these active mobile elements on the evolution of plant genomes. Analysis of the sugarcane transcriptome identified 267 clones with significant similarity to previously described plant TEs. After full cDNA sequencing, 68 sugarcane TE clones were assigned to 11 families according to their best sequence alignment against a fully characterized element. Expression was further investigated through a combined study utilizing electronic Northerns, macroarray, transient and stable sugarcane transformation. Newly synthesized cDNA probes from flower, leaf roll, apical meristem and callus tissues confirm previous results. Callus was identified as the tissue with the highest number of TEs being expressed, revealing that tissue culture drastically induced the expression of different elements. No tissue-specific family was identified. Different representatives within a TE family displayed differential expression patterns, showing that each family presented expression in almost every tissue. Transformation experiments demonstrated that most Hopscotch clone-derived U3 regions are, indeed, active promoters, although under a strong transcriptional regulation. This is a large-scale study about the expression pattern of TEs and indicates that mobile genetic elements are transcriptionally active in the highly polyploid and complex sugarcane genome.