A sugar beet (Beta vulgaris) fosmid library from the doubled haploid accession KWS2320 encompassing 115 200 independent clones was constructed and characterized. The average insert size of the fosmid library was determined by pulsed field gel electrophoresis to be 39 kbp on average, thus representing 5.9-fold coverage of the sugar beet genome (758 Mbp). PCR screening of plate pools with primer pairs against nine sugar beet genes supported the insert size estimation. BLAST searches with 2951 fosmid end-sequences originating from 1510 clones (1536 clones attempted) revealed little contamination with organellar DNA (2.1% chloroplast DNA, 0.3% mitochondrial DNA). The sugar beet fosmid library will be integrated in the presently ongoing efforts to determine the sequence of the sugar beet genome. Fosmids will be publicly available in the format of plate pools and individual clones.