The recent years have seen significant advances in both molecular biology, allowing the identification of genes and pathways related to disease, and imaging technologies that allow for improved spatial and temporal resolution, enhanced sensitivity, better depth penetration, improved image processing, and beneficial combinations of different imaging modalities. These advances have led to a paradigm shift in the scope of diagnostic imaging. The traditional role of radiological diagnostic imaging is to define gross anatomy and structure in order to detect pathological abnormalities. Available contrast agents are mostly non-specific and can be used to image physiological processes such as changes in blood volume, flow, and perfusion but not to demonstrate pathological alterations at molecular levels. However, alterations at the anatomical-morphological level are relatively late manifestations of underlying molecular changes. Using molecular probes or markers that bind specifically to molecular targets allows for the non-invasive visualization and quantitation of biological processes such as gene expression, apoptosis, or angiogenesis at the molecular level within intact living organisms. This rapidly evolving, multidisciplinary approach, referred to as molecular imaging, promises to enable early diagnosis, can provide improved classification of stage and severity of disease, an objective assessment of treatment efficacy, and a reliable prognosis. Furthermore, molecular imaging is an important tool for the evaluation of physiological and pathophysiological processes, and for the development of new therapies. This article comprises a review of current technologies of molecular imaging, describes the development of contrast agents and various imaging modalities, new applications in specific disease models, and potential future developments.