Background: Linkage analyses strongly suggest a number of QTL for production, health and conformation traits in the middle part of bovine chromosome 6 (BTA6). The identification of the molecular background underlying the genetic variation at the QTL and subsequent functional studies require a well-annotated gene sequence map of the critical QTL intervals. To complete the sequence map of the defined subchromosomal regions on BTA6 poorly covered with comparative gene information, we focused on targeted isolation of transcribed sequences from bovine bacterial artificial chromosome (BAC) clones mapped to the QTL intervals.Results: Using the method of exon trapping, 92 unique exon trapping sequences (ETS) were discovered in a chromosomal region of poor gene coverage. Sequence identity to the current NCBI sequence assembly for BTA6 was detected for 91% of unique ETS. Comparative sequence similarity search revealed that 11% of the isolated ETS displayed high similarity to genomic sequences located on the syntenic chromosomes of the human and mouse reference genome assemblies. Nearly a third of the ETS identified similar equivalent sequences in genomic sequence scaffolds from the alternative Celera-based sequence assembly of the human genome. Screening gene, EST, and protein databases detected 17% of ETS with identity to known transcribed sequences. Expression analysis of a subset of the ETS showed that most ETS (84%) displayed a distinctive expression pattern in a multi-tissue panel of a lactating cow verifying their existence in the bovine transcriptome.Conclusion: The results of our study demonstrate that the exon trapping method based on region-specific BAC clones is very useful for targeted screening for novel transcripts located within a defined chromosomal region being deficiently endowed with annotated gene information. The majority of identified ETS represents unknown noncoding sequences in intergenic regions on BTA6 displaying a distinctive tissue-specific expression profile. However, their definite regulatory function has to be analyzed in further studies. The novel transcripts will add new sequence information to annotate a complete bovine genome sequence assembly, contribute to establish a detailed transcription map for targeted BTA6 regions and will also be helpful to dissect of the molecular and regulatory background of the QTL detected on BTA6.