BACKGROUND It is controversial to predict ambulation potential using neurological level or specific pattern of muscle strength in patients with spina bifida. We attempted to analyze the relationship between the mobility outcome and its influencing factors utilizing electromyography to study motor unit activity in lower limbs. A subsidiary aim was to evaluate the external anal sphincter with electromyography and to determine the relevant factors of bowel and bladder functions. METHODS Among 156 patients with spina bifida who had received electromyographic examination and were followed at the out-patient clinic between 1998 and 2000, functional assessment with Pediatric Evaluation of Disability Inventory (PEDI) was applied in 47 randomly selected patients. Other assessments included clinical neurological examination, bowel and bladder function survey, and ambulation evaluation. We correlated the parameters of electromyography with the ambulatory ability and PEDI scores. The innervation of external sphincter was compared between groups with or without neurogenic bowel or bladder dysfunction. RESULTS The innervations of hip adductor and quadriceps were found to correlate with walking ability (p < 0.01); above muscles as well as anterior tibialis, and gastrocnemius were related to PEDI scores (p < 0.01). The neurological level still manifested correlation with walking ability (p < 0.05) and PEDI scores (p < 0.01). Both denervation potentials and recruitment pattern were important parameters for ambulation and mobility prediction. Significant difference was obtained in denervation patentials of sphincter within bowel groups (p = 0.036) and bladder groups (p = 0.016). CONCLUSIONS Both traditional neurological level and specific muscle innervation exert crucial influence on walking and mobility functions. Electromyographic assessment demonstrates its contribution in prediction of functional outcome in spina bifida.