Transcriptome sequencing by high-throughput technologies provides global gene expression levels as well as gene structure information. Moreover, analysis of samples from single individuals allows the detection of allele-specific expression. We investigated the possibility of RNA-Seq analysis using single bovine blastocysts. Embryos were in vitro produced using abattoir-derived, in vitro- matured oocytes, TALP-based fertilization and KSOM embryo culture medium. On Day 7 of culture, 5 expanded blastocysts were collected and stored in RNA extraction buffer at –80°C. Total RNA was extracted from each individual embryo using the Arcturus PicoPure RNA isolation kit including DNAse treatment. Approximately 1.5 ng of high-quality total RNA was obtained per embryo. The RNA was amplified using the SPIA-based Ovation RNA-Seq kit (NuGen, San Carlos, CA, USA). After amplification, 6 μg of cDNA was obtained and directly used for library construction with the NuGen Encore NGS Library I kit. Libraries were submitted to the University of California Davis Genome Center for 40-bp single read sequencing on an Illumina GAIIx apparatus. Data analysis was performed using CLC Genomics Workbench. On average, 38 094 173 good-quality reads were produced from each sample. Removing 9 bp from the 5′ end of the sequences greatly improved read alignment. Mapping of trimmed reads to BTAU 4.0 allowing up to 2 mismatches per read resulted in 88.9 ± 0.3% of reads aligning to the genome. Using an SNP discovery algorithm, a total of 31 993 unique SNP were detected with an average of 12 530 ± 496 SNP per sample, 50% of which were heterozygous. Of the total, 45, 21, 14 and 9% were common to at least 2, 3, 4 and 5 samples, respectively. Allelic expression imbalance, defined as 75% of reads corresponding to one allele of a heterozygous SNP with coverage ≥50, was observed in 22% of SNP among those common to at least 2 samples. Mapping the reads to the transcriptome resulted in 71.8 ± 0.4% of reads aligning to genes present in Ensembl. Among those mapping to RefSeq transcripts, 64.4% corresponded to exons, 7.2% to exon-exon boundaries and 0.3% to exon-intron boundaries, with the remainder mapping to introns. An average of 9746 ± 122 genes with RPKM greater than 0.3 were detected in each sample, with 7982 genes expressed commonly among all 5 embryos. The correlation for RPKM between sample pairs was between 0.978 and 0.993. Genes known to be almost exclusively expressed by pre-implantation embryos were present, including OCT4, NANOG and CDX2 among others. Gene ontology analysis of gene groups, divided into quintiles by level of expression, indicated that the most highly expressed genes are enriched in ribosomal proteins and oxidative phosphorylation. Genes with medium-high levels of expression were enriched in structural components including organelles and the cytoskeleton. Genes at the medium level of expression represented nuclear and chromatin proteins, whereas medium-low and low were related to regulation of transcription and DNA metabolism. We conclude that RNA-Seq from a single bovine blastocyst is possible and represents a powerful tool for understanding the biology and pathologies of pre-implantation embryo development.