Clostridioides difficile is a leading cause of healthcare-associated infections and displays phenotypic heterogeneity in key processes such as toxin gene expression (virulence) and sporulation (transmission). Understanding the mechanisms that regulate C. difficile’s physiology and pathogenesis requires tools capable of resolving phenotypically distinct subpopulations in vivo. In this chapter, we present fluorescence-based reporter systems for visualizing C. difficile gene expression in situ at single-cell resolution during infection. These spectrally compatible reporters are broadly applicable for investigating the spatial and temporal dynamics of gene expression for any target gene within the context of infection. Furthermore, they can be used in competition experiments between different C. difficile strains or to visualize C. difficile alongside other gut bacterial species, enabling spatial analysis of microbe–microbe interactions.