Objective To evaluate the short-term efficacy and safety of peroral endoscopic myotomy (POEM) combined with full thickness V-shaped resection of partial lower esophageal muscle in treatment of achalasia and to develop a method for obtaining full-thickness smooth muscle samples of lower esophageal sphincter. Methods Achalasia patients who underwent endoscopic surgery at Tianjin Medical University General Hospital from February 2018 to February 2019 were randomly divided into a group of POEM combined with full thickness V-shaped resection of partial lower esophageal muscle (group A) and POEM group (group B) according to the ratio of 1∶3 by using random number table. Operation duration, intraoperative blood loss, and other complications were compared between the two groups. Eckardt score, reflux score, high-resolution esophageal manometry and esophageal emptying parameters were compared 1 and 3 months after treatment in both groups. Additionally, the size, weight and microstructure of smooth muscle samples obtained during the operations were also compared. Results A total of 57 patients were included in the study, 16 in group A, and 41 in group B, and all underwent operations successfully. No significant differences were found in operation duration or intraoperative bleeding between the two groups [87.81±13.03 min VS 82.20±18.10 min, t=1.302, P=0.201; 6.75±1.44 mL VS 7.00 (2.00) mL, U=-0.903, P=0.348]. No serious complications occurred during or after operation. Follow-ups were performed at 1 and 3 months after operation. Eckardt score [0.00 (1.00) VS 0.00 (1.00), U=-0.156, P=0.876; 0.00 (1.00) VS 0.00 (1.00), U=-0.337, P=0.736], reflux score [0.00 (0.00) VS 0.00 (0.00), U=-0.207, P=0.836; 0.00 (0.00) VS 0.00 (0.00), U=-0.207, P=0.836], lower esophageal sphincter pressure [16.00±7.00 mmHg VS 13.76±6.21 mmHg, t=1.183, P=0.242; 15.06±4.14 mmHg VS 11.00 (7.00) mmHg, U=-1.852, P=0.064](1 mmHg=0.133 kPa), 4 s integrated relaxation pressure (6.57±2.69 mmHg VS 6.82±2.22 mmHg, t=-0.364, P=0.717; 5.96±1.84 mmHg VS 6.46±1.43 mmHg, t=-1.095, P=0.278) and the height of barium in 5 min in esophageal emptying test (2.16±0.91 cm VS 2.13±0.87 cm, t=0.127, P=0.899; 2.22±0.51 cm VS 2.10±0.87 cm, t=0.657, P=0.514) were similar between the two groups, which were lower than those before treatment (all P<0.05). In addition, no differences were found in either group in the above indicators between the two postoperative follow-ups (all P>0.05). One patient in group A and 2 in group B reported reflux symptoms during the short-term follow-up but no drug intervention was required. The size of specimens obtained from group A was bigger [(1.32±0.55) cm × (0.58±0.17) cm × (0.18±0.02) cm VS (0.28±0.05) cm × (0.13±0.03) cm × (0.10±0.03) cm, t=5.244, P<0.001)] and their weight was heavier (0.22±0.09 g VS 0.03±0.01 g, t=7.192, P<0.001) than those of group B. Complete circular muscle, intermuscular plexus and longitudinal muscle could be observed under microscopy in group A, while only circular muscle could be observed in group B. Conclusion The safety and short-term efficacy of POEM combined with full thickness V-shaped resection of partial lower esophageal muscle in the treatment of achalasia are comparable to those of POEM, which provides ideal histopathological specimens for achalasia pathology.