Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (∼28%) and well above the predicted error of Sanger sequencing (∼2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.