Abstract The analysis of genetic structure and variability of isolated species is of critical importance in evaluating whether stochastic or human-caused factors are affecting rare species. Low genetic diversity compromises the ability of populations to evolve and reduces their chances of survival under environmental changes. Petunia secreta , a rare and endemic species, is an annual and heliophilous herb that is bee-pollinated and easily recognizable by its purple and salverform corolla. It was described as a new species of the Petunia genus in 2005. Few individuals of P. secreta have been observed in nature and little is known about this species. All the natural populations of P. secreta that were found were studied using 15 microsatellite loci, two intergenic plastid sequences and morphological traits. Statistical analysis was performed to describe the genetic diversity of this rare species and the results compared with those of more widespread and frequent Petunia species from the same geographic area to understand whether factors associated with population size could affect rare species of this genus. The results showed that despite its rarity, P. secreta presented high genetic diversity that was equivalent to or even higher than that of widespread Petunia species. It was shown that this species is divided into two evolutionary lineages, and the genetic differentiation indices between them and other congeneric species presented different patterns. The major risk to P. secreta maintenance is its rarity, suggesting the necessity of a preservation programme and more biological and evolutionary studies that handle the two evolutionary lineages independently.