We examined 40 wrists of 12 embalmed and eight fresh cadavers and defined the relative position of the flexor retinaculum to the neurovascular structure, ultrasonographic markers and safe zones by ultrasonography and anatomical dissection. Both longitudinal and transverse ultrasonographic sections clearly depicted the flexor retinaculum, neurovascular bundles, median nerve, flexor tendons and bony boundaries of the underlying joints. Topographic measurement showed [i] good correlation between the actual extent of the flexor retinaculum and the ultrasonographically determined distance between bony landmarks in all hands, and [ii] the widths and lengths of well-defined safe zones. A comparison study confirmed the accuracy of ultrasonography. We conclude that these ultrasonographic landmarks can locate the flexor retinaculum and facilitate safe and complete carpal tunnel release with open or minimally invasive techniques.