Myxobacteria are widely distributed in soil and oceanic sediment with a phylogeographic separation at high levels of classification. However, it is unclear whether freshwater environments, from which there has been no isolation report of myxobacteria since 1981, are habitats for myxobacteria. In this study, we investigated the presence of myxobacteria in lake mud using a two-step strategy. First, we constructed two universal bacterial libraries from the V3–V4 (V34) and V6–V8 (V678) hypervariable regions of 16S rRNA gene sequences. High-throughput 454 pyrosequencing revealed that myxobacteria were one of the major bacterial groups in the lake mud. They accounted for 5.77% of the total sequences and 7.52% of the total operational taxonomic units (OTUs) at a phylogenetic distance of 0.03. The community composition and taxonomic structure of the mud myxobacterial community were further analysed using myxobacteria-enriched libraries targeting the V34 and V678 regions, which were amplified with Cystobacterineae- and Sorangineae-specific primer pairs respectively. Phylogenetic analysis showed that the limnetic myxobacteria exhibited closer relationships to their soil than their marine relatives, but there were also exclusive taxa of limnetic myxobacteria detected. These results, together with a survey on available GenBank data, indicate that lake mud is a primary habitat for myxobacteria.