The mitochondrial genomes are generally considered non-recombining and homoplasmic in nature. However, our previous study provided the first evidence of extensive and stable mitochondrial heteroplasmy in natural populations of the basidiomycete fungus Thelephora ganbajun from Yunnan province, China. The heteroplasmy was characterized by the presence of two types of introns residing at adjacent but different sites in the cytochrome oxidase subunits I (cox1) gene within an individual strain. However, the frequencies of these two introns among isolates from different geographical populations and the implications for the genetic structure in natural populations have not been investigated. In this study, we analyzed DNA sequence variation at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster among 489 specimens from 30 geographic locations from Yunnan and compared that variation with distribution patterns of the two signature introns in the cox1 gene that are indicative of heteroplasmy in this species. In our samples, evidence for gene flow, abundant genetic diversity, and genotypic uniqueness among geographic samples in Yunnan were revealed by ITS sequence variation. While there was insignificant positive correlation between geographic distance and genetic differentiation among the geographic samples based on ITS sequences, a moderate significant correlation was found between ITS sequence variation, geographical distance of sampling sites, and distribution patterns of the two heteroplasmic introns in the cox1 gene. Interestingly, there was a significantly negative correlation between the copy numbers of the two co-existing introns. We discussed the implications of our results for a better understanding of the spread of stable mitochondrial heteroplasmy, mito-nuclear interactions, and conservation of this important gourmet mushroom.