Pili are essential adhesive determinants for many bacterial pathogens. A suppressor mutation screen that takes advantage of a pilus-mediated self-aggregative “hockey-puck” colony phenotype was designed to identify novel regulators of type I pili in Serratia marcescens. Mutations that decreased pilus biosynthesis mapped to the fimABCD operon; to the genes alaT, fkpA, and oxyR; upstream of the flagellar master regulator operon flhDC; and to an uncharacterized gene encoding a predicted DUF1401 domain. biofilm formation and pilus-dependent agglutination assays were used to characterize the relative importance of the identified genes in pilus biosynthesis. Additional mutagenic or complementation analysis was used to verify the role of candidate genes in pilus biosynthesis. Presented data support a model that CRP negatively regulates pilus biosynthesis through increased expression of flhDC and decreased expression of oxyR. Further studies are warranted to determine the mechanism by which these genes mediate pilus biosynthesis or function.