The majority of phenotypic divergence within and between species has been proposed to be as a result of enhancer evolution; however, the exact sequences that cause this divergence remain unknown. Furthermore, mutation in Transcription Factor Binding Sites (TFBS) within enhancers is associated with many diseases and development disorders. These enhancers can be difficult to identify as they are often located up to 1 megabase from their target gene and most do not display significantly increased overall sequence conservation compared to surrounding sequences. Our approach made use of extensive chromatin data available for embryonic stem (ES) cells of mouse, human and pig origin. To identify enhancers Multiple Transcription factor bound Loci (MTL) were identified using ChIP-seq data describing the binding of nine transcription factors (TFs) in mouse ES cells. MTL regions bound by increasing numbers of TFs (MTL2-4 and MTL≥5) were found to have increased enrichment for enhancer features, histone H3 K27 acetylation (H3K27ac) and EP300, and were associated with higher expression of nearby genes. Liftover of MTL2-4 and MTL≥5 to the human and pig genomes and comparison to ChIP-seq data from human and pig ES cells identified 432 regions with conserved enhancer features in at least two species. Enhancer function was confirmed by luciferase reporter assays for a subset of these regions. TFBS were identified by Clover analysis of this set of multi-species enhancers using profiles from the JASPAR database. This analysis revealed TFBS which were highly overrepresented for both mouse and human sequences and not previously linked to pluripotency maintenance. Multi-sequence alignment (MSA) was performed for the 432 multi-species enhancers with the corresponding sequences from the human, mouse and pig genomes, revealing important characteristics of TFBS evolution. The binding site for OCT4:SOX2 dimers was found to be most highly conserved among the 3 species with 57% of TFBS being nearly identical (>90%) in all three species. Additional important ES cell expressed TFs including: KLF4, ESRRB, STAT3 and TCFCP2L1 were found to be less well conserved with less than 22% being nearly identical in all three species. Although ES cells have an extensively characterized regulatory network, our multi-species approach identified novel TFs which may be involved in pluripotency maintenance. Furthermore, this approach could be applied to additional cell types which have a less well characterized regulatory network to discover novel regulators.