New native pine forests provide potential new habitat for colonisation by stipitate hydnoid fungi but their ability to colonise and persist in these areas is unknown. Soil containing Scots pine seedlings and inoculum of Hydnellum peckii was transferred to Darroch Wids, which is a newly planted native forest on former agricultural land. Both H. peckii and Phellodon tomentosus were transferred to White Bridge, which once would have supported native Scots pine forest. Below-ground persistence of the inoculum was monitored by amplification of ITS sequences from soil DNA and RNA using species-specific primers, and root systems were screened for the presence of ectomycorrhizas of both target species. H. peckii DNA was consistently detected over 2.5 y in soil taken from the Darroch Wids site. The number of detections of H. peckii and P. tomentosus DNA at White Bridge showed a sharp decline over 1 y. RNA of the target species was detected in the majority of samples, however, no ectomycorrhizas were found at White Bridge. This suggests that establishment of new colonies of H. peckii and P. tomentosus is unpredictable.