Abstract Heritable variation in a gene’s expression arises from mutations impacting cis - and trans -acting components of its regulatory network, with expression variation often derived from trans -regulatory mutations within species. Here, we investigate how trans -regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations with trans -regulatory effects on expression of the same focal gene in Saccharomyces cerevisiae . Relative to 1766 mutations without effects on expression of this focal gene, we found that these trans -regulatory mutations were enriched in coding sequences of transcription factors previously predicted to regulate expression of the focal gene. However, over 90% of the trans -regulatory mutations identified mapped to other types of genes involved in diverse biological processes including chromatin state, metabolism and signal transduction. Finally, we find that the genomic distribution of these new regulatory mutations significantly overlaps with the genomic distribution of trans -regulatory variants segregating within S. cerevisiae .