Contents Research on bovine embryonic stem cells (bESCs) has been hampered because bESCs are cultured in conditions that are based on information obtained from culturing mouse and human inner cell mass (ICM) cells. The aim of this study was to compare gene expression in ICM and trophectoderm (TE) cell lineages of bovine embryos and to discuss the findings relative to information available for mice and humans. We separated a high‐purity (>90%) ICM and TE from bovine blastocysts by magnetic‐activated cell sorting and analysed their transcriptomes by single cell RNA‐seq. Differentially expressed genes (DEGs) were assessed using Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases. Finally, qRT‐PCR was performed to validate the RNA‐seq results. From 207 DEGs identified (adjusted p ≤ .05; fold change ≥2), 159 and 48 had greater expression in the ICM and TE cells respectively. We validated 27 genes using qRT‐PCR and found their expression patterns were mostly similar to those of RNA‐seq, including 12 novel ICM‐dominant (HNF4A, CCL24, FGFR4, IFITM3, PTCHD2, GJB5, FN1, KLK7, PRDM14, GRP, FGF19 and GCM1) and two novel TE‐dominant (SLC10A1 and WNT4) genes. Bioinformatics analysis showed that these DEGs are involved in many important pathways, such as MAPK and cancer cell pathways, and these pathways have been shown to play essential roles in mouse and human ESCs in the self‐renewal and pluripotent maintenance. As a conclusion, there were sufficient differences to allow us to conclude that the control of pluripotency in bovine ICM cells is species‐specific.