Twins, especially second-born twins, have more unfavorable pregnancy results than singletons. Despite the fact that more than half of twins have been prematurely born, research on auditory brainstem maturation in premature twins is limited. Thus, the goal of this research was to compare the maturation of the maturation of the auditory brainstem in preterm twins and singletons, as well as investigate this maturation process with regard to birth order. The present study retrospectively assessed medical files of 40 preterm twins across 20 twin pregnancies and 20 singleton infants in the Shafiezade Children's Hospital of Babol University of Medical Sciences. We incorporated infants delivered amid March 2021 and September 2021 who had a gestational age (GA) of 36 or 37 weeks, a weight larger than 1,500 g, along with passed transient evoked otoacoustic emissions (TEOAEs). All incorporated sample were examined via diagnostic auditory brainstem response (ABR) at 50 and 30 dBnHL. There were insufficiently significant variations between preterm twins and the singletons with regard to mean III and V latencies as well as III-V intervals. Furthermore, comparing mean III and V latencies as well as III-V interval values of preterm second-born twins against preterm singletons showed no discernible effect of birth order. In current retrospective research, First- and second-born twins showed the same central auditory system maturation at the brainstem level as preterm singletons.