Gerbera delavayi (fireweed, Asteraceae) is a perennial herb native to southwest China and the neighboring Vietnam. In recent years, the species' distribution and abundance have seriously declined, because its leaves have been excessively harvested for textile industry. In this study, 19 polymorphic and neutral microsatellite primer pairs were developed based on transcriptome sequencing of G. delavayi and then used to infer the influence of human activities on the genetic diversity and population structure of nine wild populations (204 individuals). Moderate genetic diversity was detected based on the allelic richness A_R = 3.436, Shannon's information index I = 0.789, observed heterozygosity H_O = 0.304, and expected heterozygosity H_E = 0.415, and moderate genetic differentiation F_ST = 0.110 (p < 0.001) was found among the nine populations. High genetic differentiation, limited gene flow, and a clear genetic barrier were detected between one population and the remaining eight. The results suggest that isolation barriers and human overexploitation have led to the moderate genetic diversity in the populations of G. delavayi. Environmental factors, floristic composition and geographic barriers of mountain ranges affected the genetic structure of G. delavayi. When utilizing the current wild resources, protection of genetic diversity both in situ and ex situ need to be considered.