Myofibrillar myopathy (MFM) encompasses a genetically heterogeneous group of human diseases caused by mutations in genes coding for structural proteins of muscle. Mutations in the intermediate filament (IF) protein desmin (DES), a major cytoskeletal component of myocytes, lead to severe forms of “desminopathy,” which affects cardiac, skeletal, and smooth muscle. Most mutations described reside in the central α‐helical rod domain of desmin. Here we report three novel mutations—c.1325C>T (p.T442I), c.1360C>T (p.R454W), and c.1379G>T (p.S460I)—located in desmin's non‐α‐helical carboxy‐terminal “tail” domain. We have investigated the impact of these and four—c.1237G>A (p.E413K), c.1346A>C (p.K449T), c.1353C>G (p.I451M), and c.1405G>A (p.V469M)—previously described “tail” mutations on in vitro filament formation and on the generation of ordered cytoskeletal arrays in transfected myoblasts. Although all but two mutants (p.E413K, p.R454W) assembled into IFs in vitro and all except p.E413K were incorporated into IF arrays in transfected C2C12 cells, filament properties differed significantly from wild‐type desmin as revealed by viscometric assembly assays. Most notably, when coassembled with wild‐type desmin, these mutants revealed a severe disturbance of filament‐formation competence and filament–filament interactions, indicating an inherent incompatibility of mutant and wild‐type protein to form mixed filaments. The various clinical phenotypes observed may reflect altered interactions of desmin's tail domain with different components of the myoblast cytoskeleton leading to diminished biomechanical properties and/or altered metabolism of the individual myocyte. Our in vitro assembly regimen proved to be a very sensible tool to detect if a particular desmin mutation is able to cause filament abnormalities. Hum Mutat 28(4), 374–386, 2007. Published 2007 Wiley‐Liss, Inc.