Traditional two-dimensional (2D) histology, while foundational in anatomic pathology for understanding the complex composition and microarchitecture of biological tissues, suffers from inherent limitations. This method is time-consuming, prone to artifacts, and fails to capture the intricate 3D tissue architecture, thereby limiting volumetric analysis and accurate quantification of diverse tissues. These limitations restrict our understanding of complex biological systems, particularly in the field of musculoskeletal research. X-ray micro-computed tomography (μCT) emerges as a promising alternative for 3D visualization of heterogeneous tissues, offering complete three-dimensional (3D) information, high spatial resolution, extensive field of view, and reduced sample preparation workload. Initially limited to mineralized tissues due to the weak X-ray absorbing properties of nonmineralized or soft biological tissues, the application of μCT has been revolutionized by the development of radiopaque contrast-enhancing staining agents (CESAs) and X-ray imaging techniques. These advancements enable high-resolution 3D visualization of both mineralized and soft tissues, offering a comprehensive view of tissue microarchitecture and quantitative virtual 3D anatomical pathology. This chapter focuses on contrast-enhanced μCT (CE-μCT) ex vivo imaging for 3D anatomical pathology of musculoskeletal tissues. We review key studies that showcase the efficacy of CESAs and discuss innovative techniques, such as chemical drying and phase contrast μCT, highlighting their applications in musculoskeletal tissues. We believe that integrating CE-μCT imaging techniques provides a comprehensive approach to studying the 3D anatomical pathology of musculoskeletal tissues, offering significant improvements over traditional 2D histopathology methods.