Myxobacteria have potential application value in developing new antibiotics and environmental protection. In this study, in order to establish a more suitable method for diversity studies of myxobacteria, the effects of primers, polymerase chain reaction (PCR) approaches and sample preservation methods on the results were compared by Illumina high-throughput sequencing. The results showed that the relative abundance and operational taxonomic unit (OTU) ratio of myxobacteria amplified by the universal primers accounted for 0.91–1.85% and 2.82–4.10% of total bacteria, indicating that myxobacteria were the dominant bacteria both in population and species numbers. The relative abundance and OTU number and ratio of myxobacteria amplified by the myxobacteria semi-specific primers were significantly higher than those amplified by the universal primers, of which the primer pair W2/802R specifically amplified myxobacteria of suborder Cystobacterineae, while the primer pair W5/802R mainly amplified myxobacteria of suborder Sorangineae and also amplified more species of suborder Nannocystineae at the same time. Among three PCR approaches, the relative abundance and OTU ratio of myxobacteria amplified by the touch-down PCR were the highest. More myxobacterial OTUs were detected in most dried samples. In conclusion, the combination of the myxobacteria semi-specific primer pairs W2/802R and W5/802R, touch-down PCR, and dry preservation of samples were more conducive to diversity studies of myxobacteria. •The order of Myxococcales was the dominant bacteria in the soil samples.•The primers had the greatest influence on diversity studies of myxobacteria.•The primers W2/802R and W5/802R were suitable for diversity studies of myxobacteria.•Touch-down PCR was conducive to diversity studies of myxobacteria.•Sample preservation by air drying was helpful to diversity studies of myxobacteria.