During the past decade, the field of mass spectrometry imaging (MSI) has greatly evolved, to a point where it has now been fully integrated by most vendors as an optional or dedicated platform that can be purchased with their instruments. However, the technology is not mature and multiple research groups in both academia and industry are still very actively studying the fundamentals of imaging techniques, adapting the technology to new ionization sources, and developing new applications. As a result, there important varieties of data file formats used to store mass spectrometry imaging data and, concurrent to the development of MSi, collaborative efforts have been undertaken to introduce common imaging data file formats. However, few free software packages to read and analyze files of these different formats are readily available. We introduce here MSiReader, a free open source application to read and analyze high resolution MSI data from the most common MSi data formats. The application is built on the Matlab platform (Mathworks, Natick, MA, USA) and includes a large selection of data analysis tools and features. People who are unfamiliar with the Matlab language will have little difficult navigating the user-friendly interface, and users with Matlab programming experience can adapt and customize MSiReader for their own needs.