The identification of DNA G-quadruplexes (G4s) in the genome is important to study different biological processes in which these structures play a role, such as genome rearrangement, transcriptional regulation and DNA replication. G4-seq allowed the high-throughput experimental mapping of G-quadruplexes in the human genome. We developed here an improved version of this method, named G4-seq2, which we applied to generate G-quadruplexes genomic maps for 12 species, selected as important models organism to study development or as pathogens of clinical relevance. Those multi-species maps, publicly available for the community, will allow to further understand the design principle of G-quadruplex formation in genomic context, to study G-quadruplex biology in those model organisms, to predict ligand targeting for therapeutic usage and to design G-quadruplex computational predictors based on genome-wide experimental measurements. Overall design: 24 library samples, 150 base pairs custom protocol (G4-Seq2) sequenced as two-times single-end reads on HiSeq 2500: 12 samples from different species for Li+ (Read-1) and K+ (Read-2) 12 samples from different species for Li+ (Read-1) and PDS+K+ (Read-2).