UNLABELLED: Myxococcota is a phylum of sterol-producing bacteria. They exhibit a clade depth for sterol biosynthesis unparalleled in the bacterial domain and produce sterols of a biosynthetic complexity that rivals eukaryotes. Additionally, the sterol biosynthesis pathways found in this phylum have been proposed as a potential source for sterol biosynthesis in the last eukaryotic common ancestor, lending evolutionary importance to our understanding of this pathway in Myxococcota. However, sterol production has only been characterized in a few species, and outstanding questions about the evolutionary history of this pathway remain. Here, we identify two myxobacteria, IMPORTANCE: Sterols are essential and ubiquitous lipids in eukaryotes, but their significance in bacteria is less understood. Sterol production in Myxococcota, a phylum of developmentally complex predatory bacteria, has provided insight into novel sterol biochemistry and prompted discussion regarding the evolution of this pathway within both the eukaryotic and bacterial domains. Here, we characterize cholesterol biosynthesis in two myxobacteria, providing evidence for distinct pathway organization and identifying a unique protein responsible for C-24 reduction. We couple these results with the phylogenomic analysis of sterol biosynthesis within Myxococcota, revealing a complicated evolutionary history marked by vertical and horizontal transfer. This suggests a mosaic acquisition of this pathway in Myxococcota and highlights the complex role myxobacteria may have had in sterol transfer to eukaryotes.