The evolution of the elastic constants, C33, C44 and C55, Poisson’s ratio and acoustic birefringence of a Fe-0.5 wt% C-21.5 wt% Mn twinning-induced plasticity (TWIP) steel with reduction by cold rolling and recrystallization annealing was assessed from measurements of the times of flight of ultrasonic waves propagating along the thickness of the rolled plates. As the reduction increased, changes in the elastic constants resulted in a steadily increasing orthotropy, which was clearly shown by Poisson’s ratio and acoustic birefringence. Although optical metallography and hardness measurements showed that partial or full recrystallization is attained after annealing at 600 °C and 700 °C, the ultrasonic measurements revealed that a high level of orthotropy remains.