MLO is a family of transmembrane proteins in land plants that play an important role in plant immunity and host-pathogen interactions, as well as wide range of development processes. Understanding the evolutionary history of MLO proteins is important for understanding plant physiology and health. In the present work, we conducted phylogenetic analysis on a large set of MLO protein sequences from publicly available databases, specifically emphasising MLOs from tomato and related species. As a result, 4886 protein sequences were identified and used to construct the phylogenetic tree. In comparison with previous findings, we identified nine phylogenetic clades, revealing the internal structure of clades I and II as additional clades, and showed the presence of monocotyledon species in all MLO clades. We identified a set of 19 protein motifs, allowing the identification of particular clades. Sixteen SlMLO proteins from tomato were located in the phylogenetic tree and identified in relation to homologous sequences from other Solanaceae species. The obtained results may be useful for further work on the use of MLO proteins in the study of mildew resistance in Solanaceae and other plant families.