Phase diversity (PD) is a powerful technique for estimating wavefront aberrations from two-dimensional images of extended scenes. PD can work with extended incoherent images and, in an adaptive optics system, does not need extra hardware in addition to the deformable mirror. For these reasons, PD should be well suited to aberration measurement in microscopy applications. But, in biological widefield microscopy, the objects being imaged are frequently three-dimensional, and the images contain out-of-focus light. In this paper, we introduce multiplane PD and show that PD can be extended to widefield imaging of three-dimensional objects. This should be particularly useful in the field of biological fluorescence microscopy where the objects are very light sensitive and the aberrations cannot easily be determined.