Aedes aegypti is the major epidemic vector of dengue outbreaks in the world. Here, a 359 bp region of the mitochondrial NADH hydrogenase subunit 4 (ND4) gene was taken as the candidate locus for study purposes from 27 Aedes aegypti mosquito larvae collected from an inland mountain area (Badulla), and two distantly located coastal areas (Hambantota and Trincomalee) in Sri Lanka. Eleven parsimonious informative sites were observed, and 7 haplotypes were identified from the 27 samples. The coastal populations displayed higher genetic diversity compared to those from the inland mountain-contained region. The phylogenetic analyses of Ae. aegypti from Badulla, Hambantota and Trincomalee including published Sri Lankan and global samples (a total of 106 sequences) revealed two separate clades: a basal clade and a derived clade. Eighty percent of the samples found from Badulla, were of the basal haplotype (H1) clustered together with West African (Senegal) samples in close proximity to the outgroup. On the contrary, Hambantota and Trincomalee samples were found dispersed in both the basal and derived clades clustering closely with samples from Mexico, Brazil, Kenya, USA and Myanmar indicating their close genetic relationship to many New World, East African and Asian varieties. Taken together, our study proposes that colonization of Ae aegypti in Sri Lanka occurred through multiple founder events (ancient multiple invasions in coastal sites and a more recent West African invasion to the inland mountain-demarcated Badulla region) and continue to exist in mutational drift equilibrium following the settlement event.