Fragmentation is the third cause of the biodiversity declination. Population genetic studies using Lepidoptera as the model species in the context of loss of habitat are scarce, particularly for tropical areas. We chose a widespread butterfly from Mexico as the model species to explore how changes of habitat characteristics (undisturbed forest, anthropogenic disturbances, and coastal areas), and climatic conditions affect genetic diversity and population structure. The Nymphalidae Eunica tatila is a common species in the Yucatan Peninsula considered to be a bio-indicator of undisturbed tropical forest, with migratory potential and a possible sex-biased dispersal. We genotyped 323 individuals collected in eight undisturbed areas, using four Inter Simple Sequence Repeats primers. Results show a high genetic diversity and no population structure. Temperature and shrub density present a positive and significant relationship with polymorphism values. Furthermore, our results show the positive effect of surrounding forest habitat on genetic diversity, confirming that E. tatila is a bio-indicator of undisturbed tropical forest. We found evidence of sex-biased dispersal. This paper represents one of the few studies on population genetics of tropical butterfly in a fragmented landscape and is, therefore, an important step in understanding the impact of habitat fragmentation on the risk of a butterflies’ decline.