Lampreys recover locomotion following a spinal cord injury (SCI). Glutamate is necessary to initiate and control locomotion and recent data suggest a crucial role for intraspinal neurons in functional recovery following SCI. We aimed to determine whether, in lampreys, axotomized spinal glutamatergic neurons, which lose glutamate immunoreactivity immediately after SCI, recover it later on and to study the long-term evolution and anatomical recovery of the spinal glutamatergic system after SCI. We used glutamate immunoreactivity to study changes in the glutamatergic system, tract-tracing to label axotomized neurons and TUNEL labelling to study cell death. Transections of the cord were made at the level of the fifth gill. TUNEL experiments indicated that cell death is a minor contributor to the initial loss of glutamate immunoreactivity. At least some of the axotomized neurons lose glutamate immunoreactivity, survive and recover glutamate immunoreactivity 1 week post-lesion (wpl). We observed a progressive increase in the number of glutamatergic neurons/processes until an almost complete anatomical recovery at 10 wpl. Among all the glutamatergic populations, the population of cerebrospinal fluid-contacting cells is the only one that never recovers. Our results indicate that full recovery of the glutamatergic system is not necessary for the restoration of function in lampreys.