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Simple Summary: Samples from an African lion cub in the Greater Kruger National Park area (South Africa), which could not walk, were tested for a gene mutation that causes one type of muscle weakness in domestic cats. The cause of the muscle weakness is believed to be genetic, but our study showed that the mutation that is found in similarly affected domestic cats was not present in the cub. Genetic diseases are more common in inbred animal populations, so this condition needs to be further evaluated to assist in the conservation of these magnificent creatures. Polyphasic skeletal muscle degeneration, necrosis and mineralization of skeletal muscle was diagnosed in eight juvenile free-ranging lions (Panthera leo), from five different litters in the Greater Kruger National Park area that were unable to walk properly. A detailed investigation was not possible in free-ranging lions, so the cause could not be determined. The cases resembled hypokalemic polymyopathy in domestic cats with muscle weakness. A candidate-gene approach previously identified a nonsense mutation in the gene coding for the enzyme lysine-deficient 4 protein kinase (WNK4) associated with the disease in Burmese and Tonkinese cats. In this study, we sequenced all 19 exons of the gene in one case, and two control samples, to identify possible mutations that may be associated with polymyopathy in free-ranging lions. Here, no mutations were detected in any of the exons sequenced. Our findings indicate that the WNK4 gene is not a major contributor to the condition in these lions. Further studies into the pathogenesis of this condition are needed to inform conservation policies for this vulnerable, iconic African species. [ABSTRACT FROM AUTHOR]