The birefringence in a dual-hole microstructured optical fiber is numerically calculated and characterized with an optical frequency domain reflectometry (OFDR) method. Due to the asymmetric dual air holes in the cross-section, the polarized LP 01 x and LP 01 y modes propagate with different group velocities and time delays. Through a polarized coherent OFDR system in experiment, the Fresnel reflection peaks for each mode are separated in the frequency domain with their corresponding beat frequency. Thus, the group birefringence −9.68×10 −4 is calculated with a beat frequency difference of 50.03 Hz between the LP 01 x and LP 01 y modes at a 6.2 m fiber end, which is in good agreement with that of −9.54×10 −4 from the theoretical simulation. Our demonstration provides an accurate and flexible method for group birefringence characterization in microstructured optical fibers with complex cross-sectional structures.