Background: While newborn screening (NBS) can identify spinal muscular atrophy (SMA) patients pre-symptomatically, in those with more severe forms, disease onset can be rapid, leading to irreversible motor neuron loss. Onset of clinical symptoms lags behind pathologic onset of disease so biomarkers for early disease detection are needed. Objectives: To describe our experience using muscle ultrasound (MUS) as an early indicator of disease manifestation in SMA patients identified at birth. Approach: Chart review of all newborn SMA patients with 2 or 3 copy number variants (CNV) of SMN2 who were seen at our institution after Pennsylvania newborn screening was implemented on March 1, 2019. Results: 5 infants were identified, 4 with 2 CNV and 1 with 3 CNV of SMN2. All had MUS performed at initial clinic visit. Average age at first visit was 6.75 (range 2-27) days of life (DOL). 4/5 patients had negative AAV9 titers and were dosed with onasemnogene abeparvovec at an average age of 18.75 DOL (range 15-21). Three patients had a normal initial MUS but showed abnormalities on repeat MUS at a range of 2.3-11 weeks later. Follow-up MUS demonstrated signs of disease through either fasciculations and/or abnormal echogenicity. These changes ranged from very mild in a single muscle to more diffuse and moderate in severity. Changes were seen as rapidly as 16 days after initial normal MUS; one patient had a severely abnormal MUS at 10 DOL prior to treatment initiation, with frequent fasciculations in all muscles studied, and granular and streak-like echogenicity in multiple muscles. MUS in patients will be followed at 3, 6, and 12 months post-dosing. Conclusions: MUS is an effective, non-invasive tool to identify early denervation in SMA. MUS showing abnormalities due to SMA in spite of prompt initiation of treatment was demonstrated in all 4 patients with 2 CNV. These findings highlight the need for decreased time from identification of affected newborns to initiation of treatment.