A comprehensive analysis of potential G4 DNA motifs (G4Ms) in genomic regions flanking transcription start sites (TSS) was performed across 13 animal species. We found that G4Ms are significantly enriched in the transcriptional regulatory regions (TRRs) of warm-blooded animals. Further analysis of human genes in different temporal groups reveals that the enrichment is not specific to genes found only in warm-blooded species but instead exist in a wide range of genes. Our findings therefore suggest that the high prevalence of G4Ms in TRRs is extensively selected in warm-blooded animals, supporting the hypothesis that G4Ms are involved in the regulation of gene transcription.