Abstract The West Indian fruit fly, A nastrepha obliqua ( M acquart) ( D iptera: T ephritidae), is a quarantine pest in C olombia that infests economically important plants, although little is known about its population dynamics. In this study, cytochrome oxidase subunit I ( COI ) and NADH dehydrogenase subunit 6 ( ND 6) mitochondrial genes were concatenated to characterise the genetic diversity and population structure of A . obliqua , associated with two factors: (1) ecosystem differences in two geographical regions of southwestern Colombia (the inter‐Andean valley of the Cauca River and the mountain region), and (2) the host plants present in the area. Additionally, a first approach was made at understanding the species demographic history. Seven haplotypes were found with Kimura 2‐parameter (K2P) genetic distances between 0.1 and 4%. Haplotype genealogies and demographic analyses suggest that the population of A . obliqua in southwestern Colombia is the result of introductory events of multiple populations of A . obliqua . However, the data indicate that the population genetic structure could be related to the ecological differences of the two regions being studied. Significant differences were also found among the distribution of haplotype frequencies of A . obliqua with regard to the diversity of host plants. This study is the first to provide an understanding of the population dynamics of A . obliqua in C olombia, which may ultimately contribute to strategies, such as sterile insect technique (SIT), for the management of the pest.