Objective: In parallel with the increase in cesarean section (CS) rates, the incidence of isthmocele has been increasing. In this study, we aimed to evaluate the effect of four different uterine incision closure techniques (single-layer locked, double-layer locked, single-layer unlocked, and double-layer unlocked) on cesarean scar healing and isthmocele formation by transvaginal ultrasound (TVUS) and saline infusion sonography (SIS). Materials and methods: This prospective, randomized study included women who underwent the first, elective, and term cesarean section delivery at the Obstetrics and Gynecology clinic of a tertiary care center between November 2018 and November 2019. A total of 60 patients were divided into four groups including 15 patients in each. Using the TVUS and SIS, the width, depth and length of the CS defect and the thickness of the residual myometrial tissue were measured. Results: There was no significant difference in the CS defect measurements and CS defect area among the four groups (p > 0.05). The lowest rate of Grade 3 isthmocele was seen in the double-locked group (5%), while the highest rate of isthmocele was seen in the single-unlocked group (35%) (p > 0.05). Conclusion: No trend of differences was seen in the four subgroups but the sample size is not big enough to draw valid conclusions.