Toxoplasma gondii is an obligate intracellular protozoon which causes toxoplasmosis, an important zoonotic disease that is endemic worldwide. Common sources of T. gondii infection in humans are food or water contaminated with oocysts and raw or undercooked meat with cysts. In animals, common sources of infection include feed, water, or litter contaminated with oocysts. The diagnosis and molecular characterization of T. gondii infection in humans and animals is crucial due to public and veterinary health importance. Various traditional and serological methods have been used in clinical practice for toxoplasmosis diagnosis, but interpreting the results remains a challenge. Several molecular techniques have also been used for the detection and genetic characterization of T. gondii, but primarily in research settings. In this paper, we review the techniques that are currently used for the diagnosis and genetic characterization of T. gondii in humans and animals, along with their advantages and disadvantages. The techniques reviewed have laid the groundwork for the future development of more effective and precise detection and characterization of T. gondii. These advances will contribute to a better understanding of epidemiology, prevention and control of toxoplasmosis. Thus, this review would be of particular interest to clinical physicians, veterinarians and researchers.