6-Azauracil (6 AU) inhibits enzymes in nucleoside synthesis and depletes the intracellular GTP/UTP pool. Mutations in transcriptional elongation machinery, as well as mutations in a variety of other pathways, exaggerate the growth defect of cells in the presence of 6 AU. Thus, identification of mutations that render cells sensitive to 6 AU will benefit study on the basis of 6 AU-sensitive phenotype. Here we performed a genome-wide screen of a fission yeast deletion library. Of 3235 single-gene deletions, 66 mutants displayed at least 50% drop of fitness in the presence of 6 AU and 60 mutants were reported for the first time; five deletions showed synthetic decrease of fitness when combined with deletion of set3(+) , which encodes a transcriptional regulator. Genes conferring tolerance to 6 AU were enriched in various processes, especially in chromosome segregation. Accordingly, genes encoding subunits of CLRC complex and spindle pole body were over-represented. Mutants were subjected to an in vivo transcript length-dependent reporter assay to assess the potential roles of deleted genes in transcriptional elongation. As with the deletions known to affect elongation, nab2Δ, nxt1Δ, rhp18Δ, SPAC24C9.08Δ, clr3Δ and ncs1Δset3Δ mutants exhibited defects in expressing long transcripts. New 6 AU-sensitive mutants identified here will help to elucidate the mechanism of action of 6 AU in the cells. Meanwhile, our study revealed novel genes potentially involved in transcriptional elongation and provided valuable targets for transcription study.