Objective To explore the relationship between latissimus dorsi myocutaneous flap and blood supply, so as to provide a scientific basis for the re-division, transposition and transplantation of latissimus dorsi myocutaneous flap.  Methods The latissimus dorsi muscle of 48 cadavers were observed by anatomy and angiography. The clinical applications of latissimus dorsi myocutaneous flap in 31 cases were reviewed.  Results The latissimus dorsi myocutaneous flap had many sources of blood supply. The main thoracodorsal artery was distributed in the upper and outer latissimus dorsi muscle. The medial and lateral branches were separated steadily with their respective distribution areas. The inner and lower parts of latissimus dorsi muscle were supplied by intercostal and lumbar arteries. The anastomosis between them and the branches of thoracodorsal artery was obvious according to X-ray angiography. The caliber was between 320-550 μm. The blood supply of the skin superficial to the latissimus dorsi muscle was from the myocutaneous artery. But the anastomosis between the perforating branches was sparse and the caliber was small near the inner and lower parts. Myocutaneous flaps were applied for wound repair, breast reconstruction and leg defect repair after mass excision in 31 cases. Thirty cases of myocutaneous flaps survived completely post-operation. One case of myocutaneous flap had early signs of partial blood flow disturbance. After treatment, blood flow gradually improved and wound healing delayed.  Conclusion The latissimus dorsi myocutaneous flap can be divided into 3 parts according to their arterial branches and anastomotic characteristics, which can provide the vascular anatomical basis for whole or partial separation, transposition or transplantation and preservation of muscle function.