In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate limited growth are typically amplifications of the SUL1 gene which encodes the high affinity sulfate transporter, resulting in fitness increases of >35%. Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493 base segment of the gene’s promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness more than 5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally-important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1. Mutations that create new Met32 or Cbf1 binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open-reading frames. We plan to extend this methodology to study the effect of mutagenesis on other yeast promoters.