Multispectral imaging (MSI) is currently in a period of transition from its role as an exotic technique to its being offered in one form or another by all the major microscopy manufacturers. This is because it provides solutions to some of the major challenges in fluorescence‐based imaging, namely ameliorating the consequences of the presence of autofluorescence and the need to easily accommodate relatively high levels of signal multiplexing. MSI, which spectrally characterizes and computationally eliminates autofluorescence, enhances the signal‐to‐background dramatically, revealing otherwise obscured targets. While this article concentrates on examples derived from liquid‐crystal tunable filter‐based technology, the intent is to showcase the advantages of multispectral imaging in general. Some technologies used to generate multispectral images are compatible with only particular optical configurations, such as point‐scanning laser confocal microscopy. Band‐sequential approaches, such as those afforded by liquid‐crystal tunable filters (LCTFs), can be conveniently coupled with a variety of imaging modalities, which, in addition to fluorescence microscopy, include brightfield (nonfluorescent) microscopy as well as small‐animal, noninvasive in‐vivo imaging. Brightfield microscopy is the chosen format for histopathology, which relies on immunohistochemistry to provide molecularly resolved clinical information. However, in contrast to fluorescent labels, multiple chromogens, if they spatially overlap, are much harder to separate and quantitate, unless MSI approaches are used. In‐vivo imaging is a rapidly growing field with applications in basic biology, drug discovery, and clinical medicine. The sensitivity of fluorescence‐based in‐vivo imaging, as with fluorescence microscopy, can be limited by the presence of significant autofluorescence, a limitation which can be overcome through the utilization of MSI. © 2006 International Society for Analytical Cytology