Abstract Between 2020 and 2022, eight calves (6 heifers, 2 bulls) in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) experienced exercise intolerance during extended physical activity. These calves exhibited an inability to keep pace with the rest of the herd, with some eventually succumbing to physical collapse from which they did not recover. Increased creatine kinase, an indication of muscle degeneration, was confirmed in biopsy and necropsy samples. Available sire pedigrees revealed a common paternal ancestor within 2 to 4 generations in all the affected calves. Dam pedigrees were unavailable; however, the herd retained replacement females that occasionally shared common ancestors with breeding bulls. Thus, a de novo autosomal recessive mutation was hypothesized as the cause of exercise intolerance in these calves. Genomic data (100K SNP) from 6 affected calves and 715 herd mates were initially used for a whole genome-wide association study, pinpointing a significant region on chromosome 29. Whole-genome sequencing of 2 affected calves combined with 145 other sequenced cattle led to the identification of a nonsense mutation in the PYGM gene, also located on chromosome 29. The mutation, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon. The protein product of PYGM, myophosphorylase, has a crucial role in metabolizing glycogen stored within skeletal muscle to generate energy for muscle cells. As a result, the recessive genotype led to greater (P < 0.05) glycogen concentrations in affected calves (n = 2) compared with heterozygous animals (n = 3) and wild-type controls (n = 2). Immunohistochemistry and label-free quantitative proteomics analysis confirmed the absence of the PYGM protein product in skeletal muscle. Myophosphorylase is needed for efficient glycogen utilization, which is crucial for producing high-quality beef. In the 2 affected calves harvested, increased skeletal muscle glycogen persisted postmortem, resulting in increased pH and dark-cutting beef, which is associated with a negative consumer perception and economic losses to the industry. In addition to identifying the cause of exercise intolerance and welfare concerns in these cattle, this study is among the first to identify a specific genetic mutation associated with dark-cutting beef. Carriers of the mutation did not display any discernible differences in meat quality or measures of animal well-being. While cattle heterozygous for the mutation may not immediately impact the beef industry, identifying carriers is paramount for implementing proactive selection and breeding strategies to prevent the production of affected calves.