Mpox—formerly monkeypox—is a re-emerging zoonotic virus disease, with large numbers of human cases reported during multi-country outbreaks in 2022. The close similarities in clinical symptoms that Mpox shares with many orthopoxvirus (OPXV) diseases make its diagnosis challenging, requiring laboratory testing for confirmation. This review focuses on the diagnostic methods used for Mpox detection in naturally infected humans and animal reservoirs, disease prevalence and transmission, clinical symptoms and signs, and currently known host ranges. Using specific search terms, up to 2 September 2022, we identified 104 relevant original research articles and case reports from NCBI-PubMed and Google Scholar databases for inclusion in the study. Our analyses observed that molecular identification techniques are overwhelmingly being used in current diagnoses, especially real-time PCR (3982/7059 cases;n= 41 studies) and conventional PCR (430/1830 cases;n= 30 studies) approaches being most-frequently-used to diagnose Mpox cases in humans. Additionally, detection of Mpox genomes, using qPCR and/or conventional PCR coupled to genome sequencing methods, offered both reliable detection and epidemiological analyses of evolving Mpox strains; identified the emergence and transmission of a novel clade ‘hMPXV-1A’ lineage B.1 during 2022 outbreaks globally. While a few current serologic assays, such as ELISA, reported on the detection of OPXV- and Mpox-specific IgG (891/2801 cases;n= 17 studies) and IgM antibodies (241/2688 cases;n= 11 studies), hemagglutination inhibition (HI) detected Mpox antibodies in human samples (88/430 cases;n= 6 studies), most other serologic and immunographic assays used were OPXV-specific. Interestingly, virus isolation (228/1259 cases;n= 24 studies), electron microscopy (216/1226 cases;n= 18 studies), and immunohistochemistry (28/40;n= 7 studies) remain useful methods of Mpox detection in humans in select instances using clinical and tissue samples. In animals, OPXV- and Mpox-DNA and antibodies were detected in various species of nonhuman primates, rodents, shrews, opossums, a dog, and a pig. With evolving transmission dynamics of Mpox, information on reliable and rapid detection methods and clinical symptoms of disease is critical for disease management.