Understanding the distribution of genetic patterns and structure is an essential target in population genetics and, thereby, important for conservation genetics. The main aim of our study was to investigate the population genetics of Usnea subfloridana, a widespread lichenised fungus, focusing on a comparison of genetic variation of its populations amongst three geographically remote and disconnected regions, in order to determine relationships amongst environmental data, variation in lichen secondary chemistry and microsatellite data in genotyped populations. In all, 928 Usnea thalli from 17 populations were genotyped using seven specific fungal microsatellite markers. Different measures of genetic diversity (allelic richness, private allelic richness, Nei’s unbiased genetic diversity and clonal diversity) were calculated and compared between lichen populations. Our results revealed a low genetic differentiation of U. subfloridana populations amongst three distant areas in Estonia and also a high level of gene flow. The results support suggestion of the long-range vegetative dispersal of subpendulous U. subfloridana via symbiotic propagules (soralia, isidia or fragments of thalli). Our study has also provided evidence that environmental variables, including mean annual temperature and geographical longitude, shape the genetic structure of U. subfloridana populations in Estonia. Additionally, a weak but statistically significant correlation between lichen chemotypes and microsatellite allele distribution was found in genotyped specimens.