The family Macrobiotidae is one of the most speciose and diverse groups among tardigrades. Although there have been attempts to reconstruct the phylogeny of this family, the evolutionary relationships within Macrobiotidae are only superficially determined as available genetic data cover only a small fraction of this vast group. Here, we present the first extensive molecular phylogeny of the family based on four molecular markers (18S rRNA, 28Sr RNA, ITS-2 and COI) associated with detailed morphological data for the majority of taxa. The phylogenetic analysis includes nearly two hundred sequences representing more than sixty species, including sixteen taxa that have never been sequenced and/or analysed phylogenetically before. Our results recovered a new monophyletic group, comprising Macrobiotus spectabilis Thulin, 1928 and Macrobiotus grandis Richters, 1911, for which we erect a new genus, Sisubiotus gen. nov., to accommodate its evolutionary distinctiveness. The largest, so far, dataset for the family Macrobiotidae showed that the genus Xerobiotus is nested within the clade representing the genus Macrobiotus deeper than it was earlier assumed, therefore we propose to suppress Xerobiotus and transfer its species to Macrobiotus. Moreover, mapping key morphological traits onto macrobiotid phylogeny exposed complex evolution of phenotypes within the Macrobiotus hufelandi group, i.e. Macrobiotus s.s. Finally, our findings enabled a detailed revision and discussion on species compositions of the most ubiquitous tardigrade genera, species groups and species complexes, which resulted in changes of taxonomic statuses of a number of macrobiotid species. All this contributes to the reconstruction of the morphological evolution within Macrobiotidae.