Numerous QTL for a variety of phenotypic traits in dairy and beef cattle have been mapped on bovine chromosome 6 (BTA6). The complete and validated information on the molecular genome organization is an essential prerequisite for the conclusive identification of the causative sequence variation underlying the QTL. In our study we describe efforts to improve the genomic sequence map assembly of BTA6 by filling-in gaps and by suggesting sequence contig rearrangements. This is achieved by the generation and in silico mapping of BAC-end sequences (BESs) from clones containing sequences placed on our high-resolution radiation hybrid (RH) map of BTA6 onto the genome sequence map. Linking high-resolution RH mapping with in silico mapping of BESs on BTA6 enabled the detection of discrepancies in chromosomal assignments of genome sequence contigs and improved the resolution of non-conclusive assignments on the genome sequence assembly. Furthermore, 37% of BESs enabled chromosomal assignment of contigs previously unassigned. Anchoring of 66% of BESs onto HSA4 confirmed the synteny of the respective region of BTA6 including the known evolutionary breakpoints. The BESs will play an important role in the ongoing efforts to complete the sequence of the bovine genome and will also provide a source for the identification of new polymorphic sites in the genome sequence to resolve QTL-containing intervals.