Abstract: It is generally accepted that genes are regulated by the interactions between transcription factors (TFs) and their binding sites (TFBSs). Some studies have demonstrated that nucleotide variants at variable positions in TFBSs affect yeast gene regulation. Furthermore, variable positions in TFBSs in association with distinct accompanying regulatory motifs of other TFs (i.e., co-TFs) can also impact gene regulation in eukaryotes. Given that, even low-affinity TF–DNA interactions are abundant in vivo; we used both low- and high-affinity TFBSs and performed a genome-wide analysis of associations between variable positions and co-TFs. We found that, in Saccharomyces cerevisiae, approximately 14% of the variable positions in TFBSs demonstrate such associations. These associations occurred in close proximity on the same promoters (i.e., highly co-localized). Moreover, such associations were highly conserved between sensu stricto yeasts and also influenced gene expression, which were consistent with enriched functional categories.