Objective To compare the effectiveness of poly ether ether ketone (PEEK) positioning devices combined with mixed reality technology versus color doppler ultrasound guidance for the localization of vessels in anterolateral thigh perforator flap. Methods A retrospective analysis was conducted on 40 patients with tissue defects after oral cancer resection who underwent repair using the anterolateral thigh perforator flap between January 2022 and June 2023. According to the different intraoperative positioning methods of the anterolateral thigh perforator flap, they were randomly divided into PEEK group (mixed reality technology positioning with PEEK positioning device combined with CTA data) and color ultrasound group (using color ultrasound guided positioning), with 20 cases in each group. There was no significant difference in gender, age, etiology, and disease duration in the two groups (P>0.05). The number of perforator vessels actually detected in the two groups of regions of interest was recorded, and compared them with the intraoperative identified number to calculate the success rate of perforator vessels; the distance between the perforating point and the actual puncture point was measured, the operation time of the two groups of flaps was recorded. ResultsP In the PEEK group, 32 perforator vessels were identified, 34 were found by intraoperative exploration, and the success rate was 94.1% (32/34); in the color ultrasound group, 29 perforator vessels were identified, 33 were found by intraoperative exploration, and the success rate was 87.8% (29/33); there was a significant difference in the success rate of identifying perforator vessels between the two groups (P<0.05). The distance between the perforating point and the actual puncture point and the operation time in PEEK group were significantly shorter than those in color ultrasound group (P<0.05). Patients in both groups were followed up 6-30 months, with a median of 17 months; there was no significant difference in the follow-up time between the two groups (P>0.05). In the PEEK group, there was 1 case of flap necrosis at the distal edge and delayed healing after trimming and dressing change. In the color ultrasound group, there was 1 case of flap necrosis at 7 days after operation and pectoralis major myocutaneous flap was selected for repair after removal of the necrotic flap. In the rest, the flap survived and the incision healed by first intention. Donor site infection occurred in 1 case in PEEK group and healed after anti-inflammatory treatment. The maxillofacial appearance of the two groups was good, the flap was not obviously bloated, and the patients were satisfied with the repair effect. Conclusion Compared with the traditional color ultrasound positioning, the positioning marker made of PEEK combined with CTA data to locate the vessels in anterolateral thigh perforator flap has higher success rate and positioning accuracy, and the flap production time is short, which has high clinical application value.