<p dir="ltr">Romaine lettuce is a vegetable crop that is frequently contaminated with and often implicated in mass outbreaks of human-pathogenic <i>Escherichia coli</i>. To date, research has focused on the specific pathogens responsible for those outbreaks and consumer- and retail-level interventions to eliminate those pathogens or to mitigate their spread. However, in many cases, no singular food source can be identified. Food microbiomes, particularly those associated phylloplanes, are a growing interest area and may be key in identifying the conditions that allow for proliferation of pathogenic organisms like <i>E. coli</i> O157:H7. Further, examination of dominant components of the lettuce microbiome may reveal suitable candidates that can be encouraged or engineered to outcompete or indicate the presence of pathogens. Until recently, most microbiome studies focused on bacteria; less understood are the fungi of these microbiomes. Many fungi have myriad applications in the prevention and mitigation of both human and plant diseases. Thus, to effectively prevent pathogenic <i>E. coli </i>outbreaks, a fundamental understanding about the fungi that cohabit the lettuce microbiome is paramount. The most frequently isolated yeast in the romaine lettuce microbiome is an undescribed yeast, revealing the dearth of information regarding nonpathogenic fungi in food systems. First, the novel yeast <i>Sporobolomyces lactucae</i> is described using a multi-locus phylogeny through genealogical gene concordance, with a discussion on the potential ecological range. Second, the genome of <i>S. lactucae </i>strain HU9244 is assembled and annotated with transcriptomic information to help guide target gene selection for biotechnology, particularly in identifying candidates for reporter genes that may assist in the detection of <i>E. coli </i>O157:H7 via olfactory or visual cues. Third, the distribution of the yeast <i>S. lactucae</i> is determined, finding the organism in various climate types and potentially on other leafy greens as well, indicating its suitability for <i>in situ</i> detection of foodborne pathogens such as <i>E. coli</i> O157:H7 commonly found on romaine lettuce and other leafy greens.</p>