Objective: To investigate whether unilateral primary acquired nasolacrimal duct obstruction (PANDO) changes the volume and water fraction of both lacrimal glands, using three-dimensional fast spin echo (3D-FSE)-Cube-Flex images, and to identify whether the lacrimal gland is a target organ in this disease. Methods: 3D-FSE-Cube-Flex images of both lacrimal glands in 25 healthy volunteers and 31 patients with uni-lateral PANDO were retrospectively reviewed. The differences in volume and water content in the lacrimal glands between the controls, non-PANDO side, and PANDO side groups were examined. Moreover, the associ-ations between magnetic resonance imaging (MRI) parameters and disease duration were assessed with corre-lation analysis. Results: The lacrimal gland volumes were not significantly different between the PANDO and non-PANDO side groups, compared to the control group (P = 0.484). However, the gland volumes tended to be increased bilat-erally in patients with PANDO. In contrast, the gland water fractions in the PANDO and non-PANDO side groups were significantly higher than those in the control group (P = 0.009 and P = 0.014, respectively), and similar between the non-PANDO and PANDO side groups (P = 0.897). No correlation was found between the disease duration and the gland MRI parameters (volume and water fraction). Conclusions: Both lacrimal glands are affected by unilateral PANDO. A change in the water fraction of these glands appears to precede the change in volume and may be a sensitive early indicator.