Mealybugs are major pests of grapevines worldwide. They cause economic losses by lowering the cosmetic value of fruits, reducing yields, transmitting viruses and resulting in the quarantine or rejection of produce in international trade. Knowledge of the species present in a vineyard is important for the adjustment of management strategies. We surveyed and accurately characterized the mealybugs infesting vineyards in one of the main production areas of Chile; 164 mealybugs were sampled from 26 vineyards in four regions of Chile and identified by DNA sequencing for two markers (cytochrome oxidase I and internal transcribed spacer 2) and morphological examination. Pseudococcus viburni (Signoret) was the most common species, followed by Pseudococcus meridionalis Prado and Pseudococcus cribata Gonzalez. Molecular variability at the COI and ITS2 loci was observed in both P. viburni and P. cribata. A comparison of haplotypes of P. viburni worldwide provides support for a recent hypothesis that this species is native to South America, a finding with direct consequences for management. Neither Pseudococcus longispinus (Targioni & Tozzetti) nor Planococcus ficus Signoret were found.