Dark septate endophytes (DSE) are diverse facultative biotrophic ascomycetes that ubiquitously colonise plant roots and may facilitate plant growth and productivity. However, the relationship between DSE resources and medicinal plants is still unclear. Here, we investigated DSE colonisation and species composition associated with roots of 25 medicinal plants collected from Anguo Medicine Planting Site in the farmland habitat, northern China in May 2017 using morphological characteristics and ITS sequencing. Typical DSE colonisation structures were observed in the roots of 25 medicinal plants. Total DSE colonisation was in the range of 2.22–100% and was significantly and positively correlated with soil organic matter, urease, and phosphatase. Host species purely explained 80.8%, 74.0%, and 79.0% of the observed variation in hyphal, microsclerotial, and total DSE colonisation, respectively. Of the 14 DSE taxa isolated from all 25 medicinal plant roots, six species were reported here for the first time in medicinal plants. Nonmetric multidimensional scaling analysis revealed that DSE colonisation and species composition significantly differed between different plant species. The DSE colonisation and species composition were predominately affected by plant identity than by soil factors. We conclude that the dynamics of DSE in the roots of medicinal plants exhibited a highly correlated plant species pattern, which further correlated with soil nutrient availability and enzymatic activity. This research provides a basis for further understanding of the ecological functions of DSE and their roles in the promotion of yield and quality of medicinal plants in the farmland ecosystems.