Monitoring gene expression is crucial for studying the responses of gene therapy and clarifying the function of certain genes in various environments. Molecular imaging is a powerful tool for noninvasive visualization of gene expression. This chapter will summarize the current status of fluorescence and bioluminescence imaging (BLI) of gene expression in live cells and tissues, and the emphasis will be mainly on the early studies that pioneered the field. First, we will describe fluorescence imaging of gene expression with a wide variety of fluorescent proteins. Next, we will discuss the strategies for BLI of gene expression. Besides incorporating the reporter gene into the host DNA, mRNA-based BLI of gene expression will also be briefly mentioned. Lastly, the construction of dual and triple fusion reporter genes will be presented. Since no single imaging modality is perfect and sufficient to obtain all of the necessary information for a given question, a combination of multiple molecular imaging modalities can offer synergistic advantages over any modality alone. Noninvasive optical imaging of gene expression has revolutionized biomedical research and the progress made over the last decade should allow molecular imaging to play a major role in the field of gene therapy. For basic and preclinical research, optical imaging is certainly indispensable for imaging gene expression. However, for clinical imaging of gene expression, positron emission tomography (PET) holds the greatest promise.