We demonstrate a novel method to characterize structural irregularities in a highly birefringent photonic crystal fiber (HB PCF) with high accuracy based on polarization coupling by torsional acoustic wave. Birefringence variation induced by the irregularities of air-hole structure in the fiber cross-section is analyzed via the transmission spectra of the acousto-optic coupling between two orthogonal polarization modes propagating along the fiber. The estimated maximum birefringence variation of two sections in the same batch of the HB PCF is 3 x 10(-6) and 10(-5), respectively, from nominal birefringence value of 4.86 x 10(-4). The comparison between the experimental results and the numerical simulation resulted in the estimation of few tens of nm variation in the air-hole structure in the PCF. Crown Copyright (c) 2010 Published by Elsevier B.V. All rights reserved.