Prodiplosis longifila Gagn e (Diptera: Cecidomyiidae) is an insect pest that attacks various types of crops, including tomato, Solanum lycopersicum L. (Solanaceae), a vegetable with substantial economic significance worldwide. Prodiplosis longifila is a widely distributed pest in Colombia, Ecuador, and Peru, countries characterized by the presence of significant geographic barriers like the Andes Mountains. It has been reported that geographic barriers affect the dynamics and genetic differentiation of insect populations. Therefore, the aim of this study was to assess the diversity, genetic structure, and demographic history of P. longifila through the analysis of sequences within the mitochondrial region of cytochrome oxidase I (COI) and rDNA-ITS2 in 27 populations located in Colombia and Ecuador. Analyses were performed on populations distributed in three geographic groups separated by the presence of the Andes Mountains. A total of 11 haplotypes were identified with the COI gene and only one haplotype in the rDNA-ITS2 was found. Analyses of population structure and demographic history revealed that there is a structure associated with the Andes, which is reflected in an uneven distribution of the haplotype frequencies between regions, but even so, gene flow between populations was detected which produces low genetic differentiation. Because P. longifila has a short-range dispersion that determines its territorial nature, it would be expected that other factors are producing the genetic exchange between populations. We suggest that the anthropogenic effect produced by farming practices, such as the use of seedlings as seed, which may carry P. longifila larvae, cause passive dispersal of pest throughout the Andes, particularly in Colombia.