A new method of three-dimensional (3-D) analysis of sclerotised structures of monogenoids was performed by processing z-series images using 3D-Doctor. Z-series were obtained from Gomori's trichrome-stained specimens of marine and freshwater monogenoids under laser scanning confocal fluorescence microscopy. Measurements obtained from 3-D images were then compared with those from 2-D images taken from both flattened and unflattened specimens. Data comparison demonstrated that 3-D morphometry allowed avoidance of over-estimation due to deformation and the reduction of errors associated with different spatial orientations. Moreover, study of 3-D images permitted observation of morphological details that are not detectable in 2-D representations. © 2006 Australian Society for Parasitology Inc.