Specific guanine-rich sequence motifs in the human genome have considerable potential to form four-stranded structures known as G-quadruplexes or G4 DNA. The enrichment of these motifs in key chromosomal regions has suggested a functional role for the G-quadruplex structure in genomic regulation. In this work, we have examined the spectrum of nucleotide substitutions in G4 motifs, and related this spectrum to G4 prevalence. Data collected from the large repository of human SNPs indicates that the core feature of G-quadruplex motifs, 5′-GGG-3′, exhibits specific mutational patterns that preserve the potential for G4 formation. In particular, we find a genome-wide pattern in which sites that disrupt the guanine triplets are more conserved and less polymorphic than their neutral counterparts. This also holds when considering non-CpG sites only. However, the low level of polymorphisms in guanine tracts is not only confined to G4 motifs. A complete mapping of DNA three-mers at guanine polymorphisms indicated that short guanine tracts are the most under-represented sequence context at polymorphic sites. Furthermore, we provide evidence for a strand bias upstream of human genes. Here, a significantly lower rate of G4-disruptive SNPs on the non-template strand supports a higher relative influence of G4 formation on this strand during transcription.