Biomarkers are required for efficient trials of neuroprotective interventions after perinatal asphyxia. This study aimed to determine whether diffusion tensor imaging (DTI) analyzed by tract-based spatial statistics (TBSS) may be a suitable biomarker of disease and treatment effects after perinatal asphyxia in small groups of patients. We performed TBSS from DTI obtained at 3 T from eight healthy control infants, 10 untreated and 10 hypothermia-treated infants with neonatal encephalopathy. Median (range) postnatal age at scan was 1 d (1-21) in the healthy infants, 6 d (4-20) in the cooled, and 7 d (4-18) in noncooled infants. Compared with the control group, fractional anisotropy (FA) was significantly reduced not only in several white matter tracts in the noncooled infants but also in the internal capsule in the cooled group. Noncooled infants had significantly lower FA than the cooled treated infants, indicating more extensive damage, in the anterior and posterior limbs of the internal capsule, the corpus callosum, and optic radiations. We conclude that perinatal hypoxic ischemic encephalopathy is associated with widespread white matter abnormalities that are reduced by moderate hypothermia. DTI analyzed by TBSS detects this treatment effect and is therefore a qualified biomarker for the early evaluation of neuroprotective interventions.