The as-spun poly (ethylene 2, 6-naphthalene dicarboxylate) (PEN) fibers prepared at take-up velocities of 3, 5, 7 and 9km/min were annealed. The annealing was done at 200, 240, 280 and 290 °C at a fixed length and the structure and properties of the annealed fibers were investigated. The wide-angle X-ray diffraction (WAXD) measurements revealed that α and β form crystals coexist in both the as-spun and annealed fibers. The weight fractions of α and β form crystals and amorphous phase were evaluated combining the data from WAXD and density measurements. The α form crystals dominated the fiber structure when the PEN fibers prepared at low take-up velocities were annealed at low temperatures whereas the β form crystals dominated the fiber structure when the PEN fibers prepared at high take-up velocities were annealed at high temperatures. When the PEN fibers obtained at 9km/min were annealed at 290°C, the weight fraction of β modification crystals reached 49%. Amorphous birefringence of annealed fibers was also evaluated from birefringence and crystalline orientation factors considering the difference in the intrinsic birefringence of α and β form crystals. The properties of PEN fibers with high β form content can be characterized by the relatively low amorphous orientation, high density and high melting point.