This study was performed to establish the genetic variability of Aedes albopictus within Subang Jaya, Selangor, Malaysia, by using the nicotinamide adenine dinucleotide dehydrogenase 5 subunit (ND5) mitochondrial DNA (mtDNA) marker. A total of 90 samples were collected from 9 localities within an area of the Subang Jaya Municipality. Genetic variability was determined through the amplification and sequencing of a fragment of the ND5 gene. Eight distinct mtDNA haplotypes were identified. The evolutionary relationship of the local haplotypes alongside 28 reference strains was used to construct a phylogram, the analysis of which revealed low genetic differentiation in terms of both nucleotide and haplotype diversity. Bayesian method was used to infer the phylogenetic tree, revealing a unique relationship between local isolates. The study corroborates the reliability of ND5 to identify distinct lineages for polymorphism-based studies and supplements the existing body of knowledge regarding its genetic diversity. This in turn could potentially aid existing vector control strategies to help mitigate the risk and spread of the dengue virus.