We have determined the high-resolution strand-specific transcriptome of the fission yeast S. pombe under multiple growth conditions using a novel RNA-DNA hybridization mapping (HybMap) technique. HybMap uses an antibody against an RNA-DNA hybrid to detect RNA molecules hybridized to a high-density DNA oligonucleotide tiling microarray. HybMap showed exceptional dynamic range and reproducibility, and allowed us to identify strand-specific coding, noncoding and structural RNAs, as well as previously unknown RNAs conserved in distant yeast species. Notably, we found that virtually the entire euchromatic genome (including intergenics) is transcribed, with heterochromatin dampening intergenic transcription. We identified features including large numbers of condition-specific noncoding RNAs, extensive antisense transcription, new properties of antisense transcripts and induced divergent transcription. Furthermore, our HybMap data informed the efficiency and locations of RNA splicing genome-wide. Finally, we observed strand-specific transcription islands around tRNAs at heterochromatin boundaries inside centromeres. Here, we discuss these new features in terms of organism fitness and transcriptome evolution.