Bats are important reservoir hosts of emerging viruses. Recent viral outbreaks and pandemics have resulted in an increased research focus on the genetic diversity, population structure, and distribution of bat species. Lyle’s flying fox (Pteropus lylei) is widely distributed throughout central Thailand, with most colonies congregating in temples within proximity to humans. A lack of knowledge regarding the genetic connectivity among different colonies hinders the investigation of zoonotic disease epidemiology and wildlife management. In this study, we hypothesized that genetic material may be exchanged between Lyle’s flying fox colonies that live in proximity. We assessed the mitochondrial displacement loop and cytochrome b nucleotide sequences of samples collected from 94 individuals from ten colonies across different roosting sites and detected limited genetic differentiation but increased nucleotide divergence within colonies. This suggests that genetic connectivity among Lyle’s flying fox colonies has experienced frequent and recent gene flow. These findings indicate that this species has maintained demographic equilibrium in a stable population, with a slight expansion event in certain populations. These data provide insights into the dynamics of bat populations, and the genetic knowledge gained presents opportunities for the improved monitoring of bat population structure.