Prior to the commercial availability of genetic testing, muscle biopsies were often pursued as a means of clarifying neuromuscular diagnoses in the pediatric population. However, once genetic testing became more accessible, the less invasive methods of a saliva or blood test became the preferred options over a muscle biopsy. Muscle biopsies are now often reserved for cases where genetic testing is unrevealing in a suspected myopathy or muscular dystrophy. To better understand the role and utility of muscle biopsies, we conducted a retrospective review of pediatric muscle biopsies performed at UCSF over the past nine years. Between March 2016 and December 2024, 88 muscle biopsies were obtained from patients aged 18 years and younger. Of the 82 biopsies where sufficient clinical information was available for analysis, the most common symptoms leading to presentation were weakness (48%), elevated CK levels (35%), hypotonia (30%), myalgia (27%), fatigability (16%), and motor delays (12%). The most common findings on muscle biopsy were mitochondrial and metabolic abnormalities (33%), nonspecific changes (16%), neurogenic changes (13%), myofibrillar abnormalities (12%), myopathic features (12%), inflammatory changes (7%), and dystrophic changes (6%). Genetic testing alone provided the final diagnosis in 9% of cases, biopsy provided supportive evidence for an uncertain genetic diagnosis in 16% of cases, and biopsy alone provided a diagnosis in 41% of cases. Mitochondrial myopathies were diagnosed based on ultrastructural changes noted on electron microscopy in 21% of cases, and 63% of cases diagnosed as mitochondrial or metabolic myopathies on biopsy showed no abnormalities with mitochondrial stains such as SDH and COX-SDH, highlighting the greater sensitivity of electron microscopy over light microscopy in the diagnosis of mitochondrial abnormalities. Muscle biopsies still play an important role in clarifying and confirming pediatric neuromuscular diagnoses even with more accessible genetic testing.